BiP AMPylation

1 AMPylation matches BiP activity to client load in the 2

3 Running title: “Inactivating AMPylation of BiP at 518”

4 Steffen Preissler1, 3, 4, Claudia Rato1, 3, Ruming Chen1, Robin Antrobus1, Shujing Ding2,

5 Ian Fearnley3, David Ron1, 4

6 University of Cambridge, Cambridge Institute for Medical Research1, Cambridge, 7 CB2 0XY, United Kingdom 8 MRC Mitochondrial Biology Unit2, Cambridge CB2 0XY, United Kingdom

9 3These authors made an equal contribution to this study

10 4Corresponding authors

11 David Ron and Steffen Preissler 12 Cambridge Institute for Medical Research 13 University of Cambridge 14 Cambridge Biomedical Campus 15 Wellcome Trust/MRC Building 16 Hills Road 17 Cambridge CB2 0XY 18 United Kingdom 19 Phone: +44 1223 768 940 20 Email: [email protected], [email protected]

21

1 BiP AMPylation

22 Abstract:

23 The endoplasmic reticulum (ER) localized Hsp70 chaperone BiP affects protein folding

24 homeostasis and the response to ER stress. Reversible inactivating covalent modification of

25 BiP is believed to contribute to the balance between chaperones and unfolded ER ,

26 but the nature of this modification has so far been hinted at indirectly. We report that deletion

27 of FICD, a gene encoding an ER-localized AMPylating , abolished detectable

28 modification of endogenous BiP enhancing ER buffering of unfolded protein stress in

29 mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD

30 AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly

31 FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In

32 vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished

33 stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular

34 basis for covalent inactivation of BiP.

35 (149 words)

36

2 BiP AMPylation

37 Introduction:

38 Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by signal

39 transduction pathways that match the complement of chaperones and to the

40 burden of unfolded protein facing the compartment. Transcriptional activation of genes that

41 enhance the capacity of the ER to process its clients and regulated translation initiation,

42 which controls the flux of unfolded proteins into the ER, comprise an unfolded protein

43 response (UPR) vital to the well being of cells, tissues and organs (Balch et al., 2008; Walter

44 and Ron, 2011). Acting alongside this coherent UPR are rapid, activity-dependent post-

45 translational changes in the disposition of the major ER chaperone BiP. Given the dominant

46 role of BiP in protein folding homeostasis in the ER, the latter stands to have considerable

47 biological significance.

48 The UPR regulates the abundance of BiP transcriptionally (Chang et al., 1989; Kozutsumi et

49 al., 1988), but this is a latent homeostatic process manifesting over hours and days. On a

50 much shorter time scale BiP’s oligomeric state is observed to change, with fewer oligomers

51 present as levels of unfolded protein increase. The architecture of BiP oligomers is

52 consistent with their role as a rapidly accessible repository of inactive BiP that the cell may

53 draw upon in short notice to cope with rapid fluctuations in unfolded protein load (Preissler et

54 al., 2015). BiP is also subject to activity-dependent post-translational modification(s). This is

55 reflected in transfer of metabolic label from intracellular pools of tritiated adenosine and 32P

56 phosphate onto BiP, covalently modifying the protein (Carlsson and Lazarides, 1983;

57 Hendershot et al., 1988) and imparting upon it a lower isoelectric point (pI) (Carlsson and

58 Lazarides, 1983; Laitusis et al., 1999). These covalent transformation(s) of BiP correlate

59 inversely with the burden of unfolded proteins in the ER (Chambers et al., 2012; Laitusis et

60 al., 1999; Leno and Ledford, 1989) and because the modified form of BiP was under-

61 represented in complex with substrates (Hendershot et al., 1988), were deemed to reflect

62 inactivating modification(s) of the chaperone (Chambers et al., 2012).

3 BiP AMPylation

63 Despite considerable effort, the chemical nature of the modification(s) was never directly

64 ascertained. ADP-ribosylation seemed a good candidate as it fit the labeling profile

65 (comprised of adenosine and phosphate), the acidic nature of the modification and its

66 susceptibility to the effect of the ADP-ribosylation inhibitor novobiocin (Laitusis et al., 1999).

67 Early work even suggested the presence of an enzymatic activity in cell lysates that could

68 transfer a label from 32P NAD+ onto BiP in vitro (Carlsson and Lazarides, 1983); but

69 molecular characterization of this enzymatic activity proved elusive. A consensus emerged

70 in regards the region of BiP that bears the modification, as both 32P orthophosphate and 3H

71 adenosine labeling mapped consistently to a cyanogen bromide (CnBr) cleavage fragment in

72 the C-terminal substrate binding domain of BiP (Thr434 to Met541) (Chambers et al., 2012;

73 Gaut, 1997). Mutations in two residues on that fragment (Arg470 and Arg492)

74 markedly attenuated radiolabeling in vivo of FLAG-tagged BiP expressed in cells, suggesting

75 that they might be the modification sites (Chambers et al., 2012), but peptides with a mass

76 consistent with ADP-ribosylated BiP were never uncovered and the assignments thus

77 remained tentative.

78 Recently it has been reported that BiP is subject to a different modification, AMPylation,

79 which results in the formation of an phosphodiester bond between the alpha phosphate of

80 ATP and a hydroxyl side chain (with release of pyrophosphate). This modification

81 of BiP is effected by a broadly conserved, ER-localized enzyme, FICD (also known as

82 HYPE) (Ham et al., 2014; Sanyal et al., 2015). There is lack of unanimity in regards to the

83 circumstances and consequences of BiP AMPylation: the Orth lab provides evidence that

84 AMPylation is an inactivation modification, induced when client protein burden is low (Ham

85 et al., 2014), whereas the Mattoo lab suggests that it is an activating modification induced by

86 ER stress (Sanyal et al., 2015). AMPylation can explain both the metabolic labeling profile of

87 BiP (from adenosine and phosphate pools) as well as the acidity of the modified form and

88 possibly even the sensitivity to novobiocin (an ATP mimetic). However the assignment of

89 AMPylation to Ser365 or Thr366 in the binding domain of BiP (Ham et al., 2014;

4 BiP AMPylation

90 Sanyal et al., 2015) is at odds with the cyanogen bromide cleavage pattern of metabolically

91 labeled BiP, suggesting a complex scenario of multiple modifications with alternative

92 outcomes.

93 Here we report on a functional and quantitative analysis of BiP modification by FICD in vitro

94 and in vivo. Our findings fit best a parsimonious model whereby AMPylation of BiP on a

95 single residue, Thr518, is the only quantitatively significant modification of the chaperone and

96 provide clues to its functional significance.

97

5 BiP AMPylation

98 Results

99 FICD is necessary and sufficient for conversion of endogenous BiP to an acidic modified form with

100 altered mobility on native-PAGE

101 Changes in the disposition of BiP induced by manipulation of conditions in the ER are

102 conveniently tracked by native gel electrophoresis (native-PAGE) and immunoblotting

103 (Freiden et al., 1992) coupled with site-directed (Preissler et al., 2015) (Figure

104 1A). Inhibition of protein synthesis led to accumulation of a prominent high mobility species

105 tentatively named ‘B’ form, whereas depletion of ER calcium progressively drew on this pool

106 to promote the assembly of BiP oligomers (Preissler et al., 2015) (Figure 1B and C). The ‘B’

107 form of BiP was also noted for its relative resistance to cleavage in vitro by a bacterial

108 protease, SubA, which cuts BiP within a highly conserved linker sequence connecting the

109 nucleotide binding (NBD) and substrate binding domains (SBD) (Paton et al., 2006) (Figure

110 1A and D). Resistance to cleavage by SubA is also a feature of the acidic, modified form of

111 BiP (Chambers et al., 2012), suggesting that the ‘B’ form observed on native-PAGE might be

112 related to the covalently modified form of BiP (previously believed to be the ADP-ribosylated

113 form).

114 To examine the potential role of FICD in elaboration of the ‘B’ form of BiP, we inactivated

115 both alleles of FICD in hamster CHO-K1 cells by CRISPR-Cas9-mediated genome editing,

116 truncating the coding sequence N-terminal to the FIC-domain active site (Figure 2A). The

117 SubA-resistant ‘B’ form, prominent in cycloheximide-treated wildtype cells, was

118 conspicuously absent in FICD-/- cells (compare lanes 2 and 5 in Figure 2B), whilst other

119 aspects of BiP’s structural dynamism (such as oligomerization in response to ER calcium

120 depletion) remained largely unchanged. FICD inactivation also led to disappearance of the

121 acidic form of BiP, present in lysates of cycloheximide-treated wildtype cells resolved by

122 isoelectric focusing PAGE (IEF-PAGE, Figure 2C). FICD-mediated AMPylation is strongly

123 autoinhibited by intramolecular competition for ATP γ-phosphate binding by glutamate 234,

124 whose mutation to (FICDE234G) de-represses FICD, whereas replacement of

6 BiP AMPylation

125 363, which is located in the highly conserved catalytic FIC motif, with alanine (FICDH363A)

126 abolishes AMPylation activity (Engel et al., 2012). In keeping with these observations,

127 enforced overexpression of the constitutively active FICDE234G led to the re-appearance of an

128 acidic form of BiP in the FICD-/- cells (Figure 2D). Wildtype FICD did not promote BiP’s acidic

129 form, even when strongly over-expressed, suggesting the existence of robust inhibitory

130 mechanisms restraining potentially deleterious effects of protein modification (Engel et al.,

131 2012). Such tight regulation is a feature common to FIC enzymes (Garcia-Pino et al., 2014)

132 but the signals leading to derepression under physiological circumstances are unknown so

133 far.

134 The FICD-/- cells provided a convenient experimental reference against which to gage time

135 dependent changes in the abundance of modified BiP following the imposition of ER stress

136 and during its resolution in wildtype cells. A decline in ‘B’ form and an increase in

137 susceptibility to digestion by SubA were noted within two hours of exposure of cells to the

138 reversible ER stress inducing agent 2-deoxyglucose (Figure 2-figure supplement 1A-C) and

139 persisted for up to eight hours thereafter. This decline in ‘B’ form occurred in the face of a

140 marked increase in FICD mRNA (Figure 2-figure supplement 1D) (Ham et al., 2014; Sanyal

141 et al., 2015), a finding consistent with regulatory mechanisms that contravene the increase

142 in mRNA. Washout of 2-deoxyglucose was associated with progressive increase in ‘B’ form

143 and the emergence of resistance to cleavage by SubA (Figure 2-figure supplement 1A-C).

144 These changes fit well the previously observed inverse correlation between levels of ER

145 stress the abundance of modified acidic BiP (Chambers et al., 2012; Laitusis et al., 1999).

146 Together with evidence that FICD was both necessary and sufficient for elaboration of the

147 acidic, modified form of BiP on IEF-PAGE lent strong support to the notion that the ‘B’ form

148 of BiP, observed on native gels, reflects the same or a related species. The high mobility of

149 the FICD-dependent ‘B’ form and its indifference to the effects of ATP (Figure 2B, lower

150 panel) (which promotes dissociation of BiP from its client proteins in vitro) are consistent with

151 a role for the FICD-mediated modification in BiP inactivation. To examine these relationships

7 BiP AMPylation

152 in further detail we measured the effects of FICD on BiP in vitro, in a system constituted of

153 pure components.

154 Whether purified as a GST-fusion protein from bacteria or from over-expressing mammalian

155 cells, active FICDE234G promoted the ATP-dependent in vitro appearance of a ‘B’ form of

156 recombinant BiP purified from bacteria. Like the endogenous ‘B’ form, found in cells, the ‘B’

157 form constituted in vitro was also partially resistant to cleavage by SubA (Figure 3A).

158 Emergence of the ‘B’ form in vitro correlated with the acquisition of a faster migrating acidic

159 form of BiP on IEF-PAGE (Figure 3B). FICD converted nearly all the detectable BiP to a

160 single acidic form in a time-dependent manner, which, like the endogenous acidic from of

161 BiP, was also relatively resistant to cleavage by SubA (Figure 3B, lane 8). Likewise, addition

162 of purified active FICDE234G to lysates from FICD-/- cells entirely converted endogenous BiP

163 into the acidic form (Figure 3C). Of note, within the resolution of IEF-PAGE, there is no

164 evidence for heterogeneity in BiP modification [also see (Carlsson and Lazarides, 1983;

165 Laitusis et al., 1999)]. This observation is consistent either with processive modification of

166 BiP or modification occurring on a single site in any given BiP molecule.

167 FICD AMPylates BiP on Thr518 in vitro

168 FICD efficiently transferred the 32P label from the alpha phosphate of ATP onto BiP,

169 consistent with AMPylation (Figure 3D). Radiolabeled BiP too was resistant to cleavage by

170 SubA indicating that BiP radiolabeled in vitro by FICDE234G reports faithfully on the behavior

171 of endogenous BiP (Figure 3-figure supplement 1). Interestingly, cleavage of radiolabeled

172 BiP (by prolonged incubation with SubA) led to emergence of a radiolabeled substrate

173 binding domain fragment (Leu417-Leu654); the nucleotide binding domain fragment, though

174 conspicuous on the Coomassie-stained gel, was entirely devoid of label (Figure 3D and

175 Figure 3-figure supplement 1B). This in vitro labeling pattern fits well with the metabolic

176 labeling of BiP by 32P orthophosphate or 3H adenosine label donors in vivo, as label was

177 mapped to a single CnBr cleavage fragment spanning Thr434 to Met541 (Chambers et al.,

178 2012; Gaut, 1997). In contrast, FICD-mediated transfer of 32P label from ATP to BiP’s

8 BiP AMPylation

179 substrate binding domain, observed here in vitro, can not be reconciled with reported

180 AMPylation of Thr366 or Ser365 (Ham et al., 2014; Sanyal et al., 2015).

181 The migration of purified recombinant BiP on IEF and native gels suggests that most or all of

182 the protein was modified in vitro with FICDE234G, yielding homogenous preparations of

183 modified BiP, whereas BiP treated with the inactive mutant FICDE234G-H363A remained

184 unmodified (Figure 3A and 3B). These preparations were examined by electrospray

185 ionization mass spectrometry and produced molecular masses of 71,060.1 and 71,395.6 Da

186 for unmodified and modified BiP, respectively (Figure 4A and Figure 4-figure supplement 1).

187 These two species were the only proteinaceous ions detected in the samples; the trailing

188 shoulder of the ion-current traces (Figure 4-figure supplement 1A) being comprised entirely

189 of singly charged low molecular mass contaminants. The mass difference of 335 Da is

190 consistent with a single covalent modification by AMP (329.12 Da) in modified BiP. The

191 difference between the predicted mass of a molecule of AMP bound via an ester linkage

192 (329.12 Da) and the observed mass difference between the modified and unmodified BiP

193 (335 Da) is reflective of the broad peaks associated with these measurements. These

194 observations indicate a single quantitative modification of BiP by AMPylation and also fit the

195 simple pattern of modified BiP migration on the IEF-PAGE. An analogous mass

196 spectrometry experiment with endogenous BiP immunopurified from cycloheximide-treated

197 wildtype CHO-K1 cells also revealed only two molecular masses (70,538.3 Da and 70,866.1

198 Da) with a mass difference of 327.8 Da, consistent with modification of BiP by a single AMP

199 moiety in vivo. BiP from AMPylation-deficient FICD-/- cells gave rise to a single species with

200 a molecular mass (70,532.2 Da) that is similar to the predicted mass of unmodified mature

201 BiP (Figure 4-figure supplement 2).

202 To identify the AMPylation site(s), samples of unmodified BiP or BiP modified in vitro with

203 FICDE234G were treated with several proteases and the peptide digests were analyzed by

204 liquid chromatography and tandem mass spectrometry (LC-MS/MS). A unique peptide

205 (doubly charged peak of 1374.15 m/z) corresponding in mass to an AMPylation of residues

9 BiP AMPylation

206 511-532 was observed in samples digested with Arg-C, and another peptide corresponding

207 to AMPylation of residues 515-528 was detected in the Asp-N digest (Figure 4B and C).

208 Moreover, another peak (916.942+) corresponding to the unmodified BiP 511-532 fragment

209 was present in the Arg-C digests of unmodified BiP and absent from the spectrum of the

210 modified form (Figure 4C, left panels) enhancing the confidence in these assignments.

211 No masses corresponding to peptides overlapping BiP 511-532 [the region identified in the

212 Arg-C and Asp-N digests to contain AMPylation site(s)], were detected in either the

213 chymotrypsin or trypsin digests of AMPylated BiP. However, all four digests contained

214 peptides covering Ser365 and Thr366, but these were all unmodified peptides (Figure 4-figure

215 supplement 3). In contrast to the unmodified 511-532 Arg-C fragment that was absent from

216 the spectrum of the modified BiP (Figure 4C), in all four digests the relative intensity of the

217 signals from peptides encompassing Ser365 and Thr366 was undiminished by modification with

218 FICDE234G (Figure 4-figure supplement 3B); this despite evidence that all the detectable BiP

219 molecules in the sample were modified (Figure 3B and 4). The mass spectrometry-based

220 evidence for non-modification of Ser365 or Thr366 also fits with the observation that under the

221 in vitro conditions studied here, FICDE234G (using α-32P-ATP as a substrate) selectively

222 modified a BiP fragment C-terminal to the SubA cleavage site (at Leu416) with no evidence

223 for modification of the N-terminal nucleotide binding domain (Figure 3D and Figure 3-figure

224 supplement 1).

225 The LC-MS/MS fragment spectra of AMP modified peptides from Arg-C and Asp-N digests

226 obtained by high energy collisional induced dissociation (HCD) or electron transfer

227 dissociation (ETD) did not directly identify the modified residue(s). However, the specificity of

228 FICD for AMPylation of hydroxyl side chains and the overlap of modified peptides from the

229 two digests narrows the modification site to three residues: Thr518, Thr525 and Thr527. Both

230 BiPT525A and BiPT527A remained substrates for AMPylation in vitro, whereas the BiPT518A

231 mutation abolished all modification of BiP (Figure 5A and B).

10 BiP AMPylation

232 BiPT366A and BiPR470E or BiPR492E [the latter are mutations that attenuate labeling of FLAG-

233 tagged BiP in vivo by 32P orthophosphate or 3H adenosine (Chambers et al., 2012)] were all

234 modified by FICDE234G in vitro with varying efficiency, as was the substrate binding-deficient

235 mutant BiPV461F (Figure 5C). The substitution of Thr518 by either alanine or

236 moderately impaired the ability of BiP to form oligomers and slightly modified mobility of the

237 monomeric (‘A’ form) of BiP on native gels, whereas mutating Thr366 had no effect on

238 oligomerization (Figure 5D). BiPT518E could not be AMPylated in vitro (Figure 5C). Yet,

239 whether exposed to FICDE234G or not, BiPT518E resembled AMPylated BiP by its migration on

240 native-PAGE and by its resistance to SubA-mediated proteolytic cleavage (Figure 5D).

241 These observations are consistent with the bulky and negatively charged side chain of

242 glutamic acid mimicking some aspect of AMPylation at Thr518, promoting a linker-protected

243 conformation. Together these observations support Thr518 as the only FICD-mediated

244 AMPylation site in vitro and suggest that changes at Thr518 introduced by AMPylation (or

245 mutation) have significant structural and functional consequences.

246 FICD AMPylates BiP on Thr518 in vivo

247 To evaluate sites of FICD-mediated BiP modification in vivo, we took advantage of a CHO-

248 K1 cell line in which the FICD gene had been inactivated by CRISPR-Cas9-mediated gene

249 targeting (Figure 2). Wildtype and FICD-knockout CHO-K1 cells were cultured in media

250 containing arginine with stable isotopes of either light or heavy nitrogen and carbon

251 (resulting in a net mass difference of 10 Da per arginine residue) and exposed to

252 cycloheximide (to enhance FICD-dependent modification of BiP). Endogenous BiP was

253 recovered by immunoaffinity purification, digested with proteases and the resultant peptides

254 were subjected to LC-MS/MS analysis. The SILAC (stable isotope labeling with amino acids

255 in cell culture) procedure was designed to quantify relative differences in the abundance of

256 unmodified and modified species in differentially labeled cultures of cells (Figure 6A).

257 Peptides from Arg-C digests corresponding in mass to the unmodified and AMPylated

258 BiP511-532 were noted in spectra derived from both untreated and cycloheximide-treated

11 BiP AMPylation

259 wildtype cells. The unmodified species was 1.5-fold more abundant in the untreated sample,

260 whereas the AMPylated species was 1.5-fold more abundant in the cycloheximide-treated

261 sample (Figure 6B). Unmodified BiP511-532 was 2.5-fold more abundant in the cycloheximide-

262 treated FICD-knockout sample than in the cycloheximide-treated wildtype sample, whereas

263 no modified BiP511-532 was detected in the cycloheximide-treated FICD-knockout sample

264 (Figure 6C). These figures attest to a large fraction of AMPylated BiP in CHO-K1 cells

265 growing in SILAC media (especially when compared to cells growing in complete media,

266 Figure 2C); perhaps a reflection of depletion wrought by dialysis of the serum.

267 The region encompassing Ser365 and Thr366 was well represented by peptides from the Arg-

268 C digest. The quantification data showed no change in the abundance of the unmodified

269 Arg-C peptide encompassing Ser365 and Thr366 (BiP337-367, Figure 6-figure supplement 1).

270 The contrast between the consistent cycloheximide-dependent and FICD genotype-

271 dependent variation in abundance of the peptide containing Thr518 and absence of change in

272 the abundance of peptides containing Ser365 and Thr366, indicates that the latter residues are

273 unlikely to be major sites of BiP AMPylation in CHO-K1 cells.

274 The mass spectra of the intact protein indicates that FICD modifies any given BiP molecule

275 on a single site both in vitro and in vivo (Figure 4 and figure supplements) and the peptide

276 fragment analysis indicates that the modification site(s) are all encompassed by peptides

277 spanning 511-532 (Figure 6 and figure supplements). The fragmentation spectra of Arg-C

278 peptides 511-532 from both endogenous BiP AMPylated in vivo, or recombinant BiP

279 AMPylated in vitro, yielded a collection of y-ions and b-ions that included all the residues on

280 the 511-532 peptide except Thr518, whereas the latter residue was conspicuously

281 represented in the ion series from the fragmentation spectrum of unmodified endogenous

282 and recombinant BiP (Figure 6-figure supplement 2). The HCD procedure likely fragments

283 the modified peptide in an unpredictable manner, rendering it unrecognizable, however, in

284 relief, the fragmentation pattern reveals Thr518 as the only modification site of BiP, in vitro

285 and in vivo.

12 BiP AMPylation

286 AMPylation efficiency depends on BiP’s conformation

287 In vitro radiolabeling revealed that amino acid substitutions distant from the primary

288 AMPylation site (Thr518) notably influenced modification efficiency. Such point mutations

289 might have indirectly influenced the accessibility of Thr518 to FICD by altering the ability of

290 BiP proteins to interact with other molecules or by changing their overall conformation. In

291 particular, we observed stronger modification of BiPV461F and BiPT366A compared to wildtype

292 BiP (Figure 5C). The V461F mutation may enhance access of FICD to BiP simply by

293 reducing competing oligomerization interactions amongst BiP proteins. In contrast, Thr366 is

294 located in the NBD and mutations at this position may interfere with ATP binding or

295 hydrolysis and thus affect the conformation of BiP. The relationship between BiP’s

296 conformation and the efficiency with which it is modified by FICD was systematically tested

297 by analysis of a series of well-characterized BiP mutants that favor certain conformational

298 states based on their altered abilities to interact with and hydrolyze (Gaut and

299 Hendershot, 1993; Petrova et al., 2008; Wei et al., 1995).

300 BiPE201G and BiPT229A, mutants that bind ATP and undergo enhanced allosteric transition but

301 are defective in nucleotide hydrolysis (Gaut and Hendershot, 1993; Petrova et al., 2008; Wei

302 et al., 1995), exhibited enhanced AMPylation rates compared to wildtype BiP (Figure 7A). By

303 contrast, BiPT37G, which is defective in ATP hydrolysis and in the allosteric transitions upon

304 ATP binding, showed slightly reduced AMPylation, whereas mutants that are severely

305 defective in adopting the ATP-bound conformation, such as the ATP binding-deficient

306 BiPG226D or BiPADDA, which is locked in the domain-uncoupled state due to a four residue

307 substitution in its interdomain linker (Laufen et al., 1999; Preissler et al., 2015), remained

308 unmodified by FICD (Figure 7A and B). Consistent with an important role for conformation in

309 determining BiP’s ability to serve as a substrate for FICD, the isolated BiP substrate binding

310 domain (purified from bacteria as a fusion to yeast Smt3) was not a substrate for FICDE234G-

311 dependent modification (Figure 7-figure supplement 1).

13 BiP AMPylation

312 Kinetic differences in modification rates of mutant BiP molecules, suggested by the

313 radiolabeling experiments, were further confirmed by tracing the time-dependent formation

E234G 314 of the ‘B’ form of BiP following exposure to FICD (Figure 7C). The t1/2max values for in

315 vitro AMPylation of BiPT229G (4.0 minutes) and BiPE201G (2.3 minutes) were significantly

316 shorter than for wildtype BiP (10.0 minutes) (Figure 7D). ATP is a substrate for FICD,

317 nonetheless, these important kinetic differences in modification were unlikely to reflect the

318 trivial consequence of varying contributions of ATP hydrolysis (by BiP) to the availability of

319 substrate nucleotide for the AMPylation reaction. Accelerated modification was not shared

320 by the ATP hydrolysis-defective BiPT37G. Furthermore, BiP AMPylation was performed in the

321 presence of millimolar ATP, whereas the affinity of FICDE234G for nucleotide is measured in

322 the 100 nM range (Bunney et al., 2014). Together, these observations indicate that

323 modification of BiP is modulated by its conformation and BiP, in the compact ATP-bound

324 conformation, is the preferred substrate for FICD. The recently determined crystal structure

325 of human BiP substrate binding domain supports this conclusion in that the loop

326 encompassing the Thr518 AMPylation site is stabilized by six polar interactions present in the

327 apo/ADP conformation that are lost in the ATP conformation (Yang et al., 2015), freeing the

328 Thr518 side-chain to react with the active site of FICD (Figure 7-figure supplement 2)

329 Consequences of AMPylation to BiP function in vitro

330 Impaired oligomerization of AMPylated BiP and its restricted conformational flexibility

331 (reflected in resistance to cleavage by SubA) suggest that the modification affects BiP

332 function and activity. Hsp70s have low basal ATP hydrolysis rates that are stimulated by J

333 protein co-factors (Liberek et al., 1991). Therefore, we compared the basal and J protein-

334 stimulated ATPase activity of unmodified BiP and BiP modified to completion. AMPylation

335 lowered the basal ATP hydrolysis rate of BiP by ~50% (Figure 8A and B) and, while the

336 ATPase activity of unmodified BiP was enhanced by the presence of a J-domain in a

337 concentration-dependent manner, AMPylated BiP was almost entirely resistant to J-

338 mediated stimulation of ATP hydrolysis (Figure 8C).

14 BiP AMPylation

339 AMPylation barely influenced steady-state binding of substrate peptide to BiP: The

340 dissociation constants of the complex between unmodified BiP or AMPylated BiP and a well-

341 characterized substrate peptide, HTFPAVL, measured at equilibrium (in the presence of

BiP BiP-AMP 342 ADP) by fluorescence polarization, were similar (Kd 16.7 µM and Kd 11.5 µM; Figure

343 8D) and within the range previously reported (Chambers et al., 2012; Marcinowski et al.,

344 2011). However, challenge with excess unlabeled peptide revealed a markedly higher “off”

BiP 345 rate of the complex between AMPylated-BiP and the bound peptide (koff 0.037 ± 0.006

-1 BiP-AMP -1 346 min vs. koff 0.212 ± 0.021 min ; Figure 8E).

347 These observations support the view that AMPylated BiP preferentially populated an ATP-

348 like state with high substrate “on” and “off” rates, even when associated with ADP, whereas

349 in the ATP-bound state AMPylated BiP was rendered inactive by its inability to respond to J

350 protein co-factor.

351 Enhanced buffering capacity of the FICD-deficient ER

352 AMPylation weakens BiP interaction with its substrates and was therefore hypothesized to

353 reverse BiP’s ability to repress the UPR transducers - whether imposed directly by binding to

354 their regulatory lumenal domains (Bertolotti et al., 2000) or indirectly by competing for

355 unfolded protein substrates (Pincus et al., 2010), BiP repression of the UPR involves stable

356 engagement of clients in its substrate binding domain. To test the derivative prediction that

357 enforced expression of an active FICD would lead to UPR activation, wildtype FICD or a

358 catalytically-dead or active FICDE234G was introduced by transient transfection into CHO cells

359 bearing an integrated CHOP::GFP UPR reporter. The dormant reporter was activated by

360 tunicamycin-induced ER stress (Figure 9A, panels 1 and 2; a positive control), and by

361 acquisition of a constitutively active FICDE234G but not by a catalytically dead FICDE234G-H363A

362 nor by the regulated wildtype enzyme (Figure 9A, panels 3-5). The robustness of these

363 observations and the independence of UPR activation from endogenous FICD – an

364 important point given the evidence for FICD dimerization (Bunney et al., 2014) - are attested

365 to by similar observations made in FICD knockout cells (Figure 9-figure supplement 1).

15 BiP AMPylation

366 The induction of the UPR reporter in CHO-K1 cells was mirrored by the behavior of

367 endogenous BiP, whose levels increased in a time-dependent manner in human HEK 293

368 cells upon activation of a conditional allele encoding FICDE234G (Figure 9B). Qualitatively

369 similar observations were made in two separate clones of FICDE234G-expressing cells [Figure

370 9C; the conspicuous basal (Dox-independent) SubA resistance of endogenous BiP, lanes 5

371 and 7, likely reflecting basal leakiness of the expression system]. These findings are readily

372 explained by the engagement of a feedback mechanism (the UPR) that defends a level of

373 active BiP and compensates for the accumulation of inert, AMPylated BiP (‘B’ form). As

374 suggested previously (Sanyal et al., 2015), the capacity of the cells to compensate for the

375 consequences of deregulated FICD activity is limited; reflected here by the adverse effect of

376 sustained FICDE234G expression on cell viability (Figure 9D).

377 The aforementioned observations support the inactivating nature of FICD-mediated BiP

378 modification and suggest that in some circumstances cells deficient in FICD may have

379 elevated levels of active BiP. Given the strong repressive role of BiP on the UPR, enhanced

380 availability of active chaperone attendant upon the absence of FICD and the consequential

381 enhanced capacity to buffer unfolded protein stress might attenuate UPR signaling. Strong

382 feedback control operative in the UPR specifies that such attenuation, were it to occur,

383 would be transient and so more likely to be detected by monitoring early events in the UPR,

384 such as PERK and IRE1α autophosphorylation, than later events, such as reporter gene

385 activity. Furthermore, “over-chaperoning” attendant upon the absence of FICD would likely

386 be revealed by manipulating the flux of proteins into the ER of a secretory cell. These

387 theoretical considerations led us to AR42j pancreatic acinar cells - a secretory cell type in

388 which PERK and IRE1α autophosphorylation is easy to detect (Bertolotti et al., 2000).

389 As expected, FICD inactivation by CRISPR-Cas9 gene editing in AR42j cells eliminated both

390 the acidic form of BiP from IEF-PAGE and the ‘B’ form from native-PAGE (Figure 10A-C and

391 Figure 10-figure supplement 1B). Wildtype and FICD-lacking AR42j cells were exposed to

392 cycloheximide to build up pools of modified BiP (in the wildtype cells) and then to the ER

16 BiP AMPylation

393 stress-inducing agent DTT, whose ability to activate the UPR does not require ongoing

394 protein synthesis (Bertolotti et al., 2000). FICD knockout led to a conspicuous temporal

395 delay in PERK and IRE1α autophosphorylation, observed in two independently derived

396 FICD-/- AR42j clones (Figure 10D). Sluggish activation of the UPR in the FICD-/- cells was

397 also reflected in attenuated PERK-dependent repression of protein synthesis imposed by

398 DTT on cells pre-treated with cycloheximide. In this experiment wildtype or FICD-/- AR42j

399 cells were pre-exposed to cycloheximide for 3 hours followed by brief washout to allow

400 recovery of protein synthesis, which was measured by incorporation of puromycin label into

401 newly synthesized proteins. In wildtype cells, DTT led to marked attenuation of protein

402 synthesis, regardless of pre-exposure to cycloheximide (Figure 10E, compare lanes 1 and 2

403 and 3 and 4), however pre-exposure to cycloheximide attenuated subsequent translational

404 repression by DTT-mediated PERK activation in FICD-/- cells (Figure 10E, lanes 5-8).

405 The aforementioned defect in UPR activation in FICD-/- cells was both fleeting and

406 dependent on the pre-imposition of a low incoming protein flux regime on the ER (attained

407 experimentally by cycloheximide pre-treatment), as FICD deletion had no measureable

408 effect on the kinetics of CHOP::GFP expression induced by treatment with tunicamycin

409 (Figure 10-figure supplement 2). These observations are inconsistent with the role proposed

410 for FICD in signaling by the PERK branch of the UPR (Sanyal et al., 2015) and suggest

411 instead that FICD-mediated BiP inactivation adjusted the level of active BiP to transient

412 fluctuations in unfolded protein flux into the ER. In the absence of this mechanism, FICD

413 knockout cells experienced an excess of functional chaperone, which was revealed here as

414 attenuated activation of the earliest steps of the UPR.

415

17 BiP AMPylation

416 Discussion

417 Previous glimpses at BiP modification have been largely indirect: Inferred from transfer of

418 radiolabel from intracellular 32P phosphate or 3H adenosine pools to an acidic form of BiP

419 and have led to the conclusion that the modification(s) consists of or ADP-

420 ribosylation or both. Attempts to pinpoint these hypothesized modifications directly were

421 unsuccessful. The discovery of FICD-mediated BiP AMPylation (Ham et al., 2014; Sanyal et

422 al., 2015) was thus an important breakthrough whose significance is enhanced further by our

423 finding that elimination of FICD abolishes all evidence for endogenous BiP modification in

424 vivo as detected by IEF- and native-PAGE, and changes in mass of the endogenous protein.

425 These findings unify and clarify the pre-existing observations regarding BiP modification and

426 indicate that FICD-mediated AMPylation on Thr518 is the major, if not the only covalent

427 change in this chaperone detectable by the existing methods. Furthermore, FICD-mediated

428 AMPylation on Thr518 is sufficient to account for the previously observed key features of

429 modified BiP, namely its acidity and inertness.

430 Our findings are consonant with metabolic labeling experiments that mapped the

431 modification to the C-terminal substrate binding domain of BiP and provide positive evidence

432 for absence of significant FICD-mediated modification of Ser365 or Thr366, neither in vitro nor

433 in vivo. Provided ATP as a co-substrate, FICD modifies BiP in vitro at a single site with a

434 mass expected of AMPylation. AMPylation maps solely to peptides encompassing Thr518

435 and these are modified to high stoichiometry in vitro. Furthermore, an unbiased chemical

436 proteomic approach to profiling proteins that are modified by FICD in a cell lysate in vitro led

437 to the identification of BiP Thr518 as an AMPylation site, whereas no modification at Ser365 or

438 Thr366 was reported (Broncel et al., 2015). It is thus tempting to speculate that Thr518 is the

439 only quantitatively significant modification of BiP carried out by FICD in vivo too. However,

440 the discovery that an FIC-domain containing bacterial enzyme AMPylates eukaryotic targets

441 (Kinch et al., 2009; Yarbrough et al., 2009) was closely followed by the realization that the

442 domain may also participate in transfer of part of other pyrophosphate-containing

18 BiP AMPylation

443 metabolites, leading to UMPylation, mono-phosphorylation and conjugation of

444 (reviewed inGarcia-Pino et al., 2014). Thus, one must keep an open mind in

445 regards to the possibility of parallel FICD-mediated modification of BiP (and possibly other

446 ER proteins) by metabolites (other than ATP) found in the ER

447 The phenotype of FICD deletion in Drosophila is restricted to a defect in light sensing and

448 involves disruption of neurotransmitter (histamine) re-uptake via glial cells. The subcellular

449 loci of Drosophila FICD action are unclear: Consistent with an ER-based function, the

450 protein co-fractionates with BiP. However, HRP-tagged endogenous FICD led to prominent

451 staining of capitate projections, a glial cell plasma membrane component of photoreceptor

452 synapses (Rahman et al., 2012). Therefore, it is possible that FICD may have substrates

453 other than BiP that figure prominently in its action in specific cell types.

454 Thr518 of hamster BiP is highly conserved in other . The crystal structure of the

455 isolated substrate binding domain of human BiP (PDB 5E86) and yeast Kar2/BiP (PDB

456 3H0X) (locked in the ADP-like state), shows the corresponding residues - human Thr518 and

538 457 Kar2 Thr . - to be located on a loop connecting beta strands 7 and 8 (L7,8), with its side-

458 chain stabilized by a network of polar contacts with the conserved side chain of

459 Asp515/Asp535 and the backbone amine of Asn520/Lys540. However both interactions are

460 disrupted in the substrate binding domain of human BiP in the ATP state (PDB 5E84).

461 Exposure of the Thr518 side chain to the solvent is likely enhanced further by the loss of four

462 other polar interactions that stabilize L7,8 in the ADP-state (Yang et al., 2015). Therefore,

463 mobilization of the side chain by allosteric signals attendant upon ATP binding is suggested

464 to enhance exposure of Thr518 to solvent explaining the selective FICD-mediated

465 modification of ATP-bound BiP and the absence of modification of mutant variants of BiP

466 that are defective in domain coupling (BiPADDA, BiPG226D and to a lesser extent BiPT37G).

467 These recent observations on human BiP (Yang et al., 2015) also showcase the long range

468 allostery common to all Hsp70 proteins (Mayer, 2013): the identity of the nucleotide bound in

469 the nucleotide binding domain affects stability of L7,8 on the opposite side of the protein. It

19 BiP AMPylation

470 seems plausible that a modification affecting the disposition of this loop could reach the

471 nucleotide binding domain through a reciprocal set of allosteric interactions to explain the

472 effects of AMPylation on nucleotide hydrolysis and responsiveness to J proteins.

473 An earlier study from our lab revealed that mutations affecting residues R470 and R492

474 consistently diminished labeling of over-expressed mutant BiP from 32P orthophosphate or

475 3H adenosine pools in vivo (Chambers et al., 2012), leading us to suggest that these

476 residues undergo ADP-ribosylation in vivo. The current findings indicate that our earlier

477 conclusions were in error and suggest that the important structural role of R470 (in all

478 Hsp70s (Fernandez-Saiz et al., 2006)) and R492 (specifically in BiP (Yang et al., 2015)) may

479 have given rise to defective allosteric transitions by the R470 and R492 BiP mutants. Such

480 defects appear to have a more conspicuous effect on modification of over-expressed BiP in

481 vivo than on the modification of pure recombinant BiP by FICD in vitro.

482 The structures of nucleotide-bound Hsp70s also provide hints to the functional

483 consequences of BiP AMPylation. Residues comprising beta strand 8 in the substrate

484 binding domain of the ADP-bound form of BiP and DnaK are dramatically delocalized in the

485 ATP bound form (Kityk et al., 2012; Qi et al., 2013; Yang et al., 2015) enabling the domain

486 movements that underlie the functional allosteric transitions (Mayer, 2013). By altering the

487 conformation of the preceding loop, AMPylation of Thr518 could facilitate the melting of beta

488 strand 8, thus favoring a new state of the chaperone. Our observations suggest that this new

489 state would resemble the ATP-bound state in terms of high substrate “off” rates, but would

490 differ from it in terms of (un)responsiveness to J protein-driven ATP hydrolysis. It is notable

491 in this regard that the ATP-like conformation can be uncoupled from responsiveness to J

492 protein, as a mutation of BiP’s substrate binding domain (G461P/G468P) that locks the

493 protein in an ATP-like conformation is also refractory to J protein-mediated stimulation of

494 ATP hydrolysis (Yang et al., 2015).

495 These structural considerations fit with the established inverse correlation between activity of

496 BiP (imposed by the burden of client proteins) and the extent of modification: Nutrient

20 BiP AMPylation

497 deprivation and protein synthesis inhibitors, which lower the flux of unfolded proteins into the

498 ER, increase the acidic form of BiP in cultured cells (Laitusis et al., 1999; Ledford and

499 Jacobs, 1986) and animal tissues (Chambers et al., 2012), whereas imposition of ER stress

500 leads to less modified BiP (Chambers et al., 2012; Ham et al., 2014; Hendershot et al.,

501 1988; Laitusis et al., 1999; Leno and Ledford, 1989) and our observations here. All this

502 suggests a simple mechanism whereby substrate-free, ATP-associated BiP partitions

503 between two mutually exclusive fates: entering the substrate binding cycle or AMPylation.

504 The former is driven by the concentration of unfolded client proteins and catalyzed by J

505 proteins, whereas the latter imposes on BiP an inactive conformation that disfavors the J

506 protein-mediated ATP hydrolysis required for high affinity client binding (De Los Rios and

507 Barducci, 2014; Misselwitz et al., 1998). High client “off” rates ensure that AMPylated BiP

508 would neither interfere with protein folding by interacting excessively with substrates, nor

509 would it unduly repress the UPR transducers. And futile cycles of ATP hydrolysis would be

510 obviated by refractoriness to stimulation by J protein. However, given the reversibility of the

511 modification, this inert BiP would serve as a repository for active chaperone were it needed

512 (Figure 11, a model).

513 Whilst such a simple kinetic competition model could account for the inverse relationship

514 between unfolded protein load and AMPylation, there are hints of other layers of refining

515 regulation. Like other type II or III FIC domain-containing proteins, FICD is intrinsically

516 repressed by the insertion of a conserved glutamate into the ATP binding pocket,

517 delocalizing the co-substrate ATP (Bunney et al., 2014; Engel et al., 2012). Relief of such

518 repression, imposed experimentally by the E234G mutation (Engel et al., 2012) or by a

519 nanobody (Truttmann et al., 2015), is likely attained physiologically through allosteric

520 mechanisms. Evidence that FICD binds unfolded proteins directly (Sanyal et al., 2015) hint

521 at the possibility that such allostery might be responsive to the burden of unfolded proteins in

522 the ER or might be regulated by FICD’s oligomeric state or co-factor binding (Bunney et al.,

523 2014). Coupling of FICD’s intrinsic activity to protein folding homeostasis in the ER could

21 BiP AMPylation

524 conserve energy by limiting the futile cycles of AMPylation and de-AMPylation prescribed by

525 regulation based solely on kinetic competition with substrates for BiP in the ATP-bound state.

526 Furthermore, regulation of FICD enzymatic activity would explain the dissociation between

527 transcriptional induction of the FICD gene during the UPR and the emergence of modified

528 BiP, which, as shown here, is delayed until after resolution of the stress.

529 Recent findings suggest the existence of an alternative inactive state of BiP effected by

530 oligomerization. The architecture of the oligomers, whereby one BiP molecule engages the

531 inter-domain linker of another as a conventional substrate peptide (Preissler et al., 2015),

532 implies that BiP protomers are locked in the ADP-bound state and unlikely to serve as a

533 substrate for AMPylation. This conjecture fits the observation that depletion of ER calcium,

534 which promotes BiP oligomerization at the expense of substrate binding, is associated with

535 gradual depletion of the FICD-dependent modified ‘B’ form of BiP (Preissler et al., 2015).

536 The benefits of ensuring an adequate reserve of chaperones to cope with unfolded protein

537 burden are clearly revealed by the consequences of defects in UPR signaling (Walter and

538 Ron, 2011). But the emergence of active mechanisms to downregulate BiP activity suggest

539 that an excess of functional BiP might have a cost too. This cost is less obvious than that

540 associated with UPR defects, as FICD inactivation results in no conspicuous growth defect

541 in cultured cells. Nonetheless, our findings indicate that in circumstances of limited client

542 protein load, the absence of FICD shifts the equilibrium in the ER in favor chaperones over

543 their clients (at least as reflected in attenuated UPR signaling). BiP over-expression works in

544 the same direction and is known to impose a measure of inefficiency in the secretion of

545 certain proteins (Dorner et al., 1992), it will thus be interesting to learn if FICD inactivation

546 also promotes inefficiency in secretion and if so, under what circumstances.

547

22 BiP AMPylation

548 Materials and Methods:

549 Plasmid construction

550 Supplementary File 1 lists the plasmids used, their lab names, description and notes their

551 first appearance in the figures and their corresponding label, and provides a published

552 reference, where available.

553 A combination of PCR-based manipulations, restriction digests and site-directed

554 mutagenesis procedures were used to mobilize the coding sequence and produce in-frame

555 fusions with the affinity tags [GST, hexahistidine (His6) and His6-Smt3 epitopes] or mCherry

556 fluorescent marker, and to create the point mutations indicated in the text.

557 Mammalian cell culture

558 All cells were grown in tissue culture dishes or multi-well plates (Corning) at 37°C and 5%

559 CO2 and the following cell line-specific media were used:

560 CHO-K1 cells (ATCC CCL-61) were phenotypically validated as auxotrophs and their

561 Cricitulus griseus origin was confirmed by genomic sequencing. The cells were cultured in

562 Nutrient mixture F-12 Ham (Sigma) supplemented with 10% (v/v) serum (FetalClone II;

563 HyClone), 1 x Penicillin-Streptomycin (Sigma) and 2 mM L- (Sigma).

564 AR42j cells (ATCC CRL-1492) were phenotypically validated by documenting inducibility of

565 amylase expression by dexamethasone and their Rattus norvegicus origin was confirmed by

566 genomic sequencing. They were cultured in DMEM (Sigma) supplemented with 10% (v/v)

567 serum (FetalClone II, HyClone), 1 x Penicillin-Streptomycin (Sigma), 2 mM L-glutamine

568 (Sigma), 1 x non-essential amino acids (Sigma) and 50 µM β-mercaptoethanol (Gibco; Life

569 Technologies).

570 HEK293T cells (ATCC CRL-3216) were cultured in DMEM (Sigma) supplemented with 10%

571 (v/v) serum (FetalClone II; HyClone), 1 x Penicillin-Streptomycin (Sigma) and 2 mM L-

572 glutamine (Sigma). Flp-In T-REx 293 cells (Invitrogen) were cultured in the same medium

573 except that 10% (v/v) tetracycline-free serum (FBS Premium, PAN-Biotech) was used.

23 BiP AMPylation

574 All experiments with untransfected cells were performed at cell densities of 60-90%

575 confluence. For pharmacological treatments the drugs were first diluted into pre-warmed

576 culture medium, mixed and immediately applied to the cells by medium exchange. Unless

577 indicated otherwise the following final concentrations were used: 100 µg/ml cycloheximide

578 (Sigma), 0.5 µM thapsigargin (Calbiochem), 1 mM DTT, 2.5 µg/ml tunicamycin (Melford), 10

579 µg/ml puromycin (Calbiochem), and 3 mM 2-deoxy-D-glucose (ACROS Organics).

580 FICD knockout using the CRISPR-Cas9 system

581 Three single guide RNA sequences (plasmids UK1448, UK1449 and UK1450;

582 Supplementary File 1) for targeting the third exon of Cricetulus griseus (Chinese hamster)

583 FICD were selected from the CRISPy database [URL:

584 http://staff.biosustain.dtu.dk/laeb/crispy/, (Ronda et al., 2014)] and duplex DNA

585 oligonucleotides of the sequences were inserted into the pSpCas9(BB)-2A-GFP plasmid

586 (plasmid UK1359; Supplementary File 1) following published procedures (Ran et al., 2013).

587 2 x 105 CHO-K1 or CHO-K1 CHOP::GFP reporter cells (Novoa et al., 2001) were plated in 6-

588 well plates. Twenty-four hours later the cells were transfected with a combination of

589 guideRNA/Cas9 plasmids UK1448 and UK1449 or UK1448 and UK1450 (2 µg total plasmid

590 DNA per transfection) using Lipofectamine LTX (Invitrogen). Thirty-six hours after

591 transfection the cells were washed with PBS, resuspended in PBS containing 4 mM EDTA

592 and 0.5% (w/v) BSA, and GFP-positive cells were individually sorted by fluorescence-

593 activated cell sorting (FACS) into 96-well plates using a MoFlo Cell Sorter (Beckman

594 Coulter). Clones were then analysed by a PCR-based assay to detect FICD mutations as

595 described (Klampfl et al., 2013). Briefly, primers were designed for the region encompassing

596 the FICD RNA guide target sites and the reverse primer was labeled with 6-

597 carboxyfluorescein (6-FAM) on the 5’ end. A PCR reaction was set up using 5 µl of

598 AmpliTaq Gold 360 Master Mix (Applied Biosystems), 0.6 µl of a mix of 10 µM forward and

599 labeled reverse primers, 3.4 µl H2O and 1 µl genomic DNA (approximately 10 ng/µl). PCR

600 was performed as follows: 95°C for 10 minutes, 10 x (94°C for 15 seconds, 59°C for 15

24 BiP AMPylation

601 seconds, 72°C for 30 seconds), 20 x (89°C for 15 seconds, 59°C for 15 seconds, 72°C for

602 30 seconds), 72°C for 20 minutes. PCR products were diluted 1:100 in water and fragment

603 length was determined on a 3130xl Genetic Analyzer (Applied Biosystems) and the data

604 were analyzed using the Gene Mapper software (Applied Biosystems). Clones for which

605 frameshift-causing insertions or deletions were detected for both alleles were sequenced to

606 confirm the FICD mutations.

607 FICD knockouts in the AR42j cell line were created as described above, using two single

608 guide RNA sequences (plasmids UK1503 and UK1504; Supplementary File 1) for targeting

609 the third exon of Rattus norvegicus (rat) FICD selected using the CRISPR Design tool [URL:

610 http:// http://crispr.mit.edu/, (Zhang Lab)]. These cells were electroporated using the Neon

611 transfection system (Life Technologies) as described (Tsunoda et al., 2014).

612 Construction of stable cell lines expressing mutant versions of FICD

613 The effect of FICD overexpression was analyzed in mammalian cell clones carrying stable

614 transgenes that encode doxycycline-inducible mutant versions of FICD. To generate stable

615 cell lines, plasmid DNA encoding FICDE234G (UK1440) and FICDE234G-H363A (UK1446) were

616 introduced into the Flp-In T-REx 293 cell line (Invitrogen) as described by the manufacturer's

617 protocol. Briefly, HindIII-XhoI DNA fragments containing the coding sequences for FICDE234G

618 and FICDE234G-H363A were sub-cloned into pcDNA5/FRT/TO (Invitrogen). The resulting

619 expression plasmids were individually co-transfected along with the Flp-recombinase

620 expression vector pOG44 (Invitrogen) using Lipofectamine 2000 (Invitrogen) into Flp-In T-

621 REx 293 cells. Isogenic clones expressing the transgene under a doxycycline-inducible

622 promoter were selected for resistance to blasticidin (3 µg/ml; Thermo Fisher) and

623 hygromycin (250 µg/ml; Invitrogen) and sensitivity to zeocin (50 µg/ml; Invitrogen).

624 Cell proliferation assay

625 Flp-In T-REx 293 cells with stable transgenes encoding FICDE234G and FICDE234G-H363A were

626 plated at a density of 8 x 103 cells per well on a 24-well tissue culture plate and grown for 24

25 BiP AMPylation

627 hours. The cells were then treated with 0.1 µg/ml doxycycline for the indicated times.

628 Afterwards, the cells were washed once in regular medium and then maintained in regular

629 medium for three days. Following the recovery period, the medium was replaced with fresh

630 medium containing 0.02 mM WST-1 (Dojindo) and 1-methoxy phenazine methosulfate

631 (Sigma), and the cells incubated for 60 minutes at 37°C before absorbance was measured

632 at 440 nm. Each experiment was performed in duplicates and repeated three times.

633 Flow cytometry

634 The effect of FICD overexpression (wildtype, FICDE234G and FICDE234G-H363A mutant versions)

635 on the unfolded protein response was studied by transient transfection of CHO-K1 wildtype

636 and FICD-/- CHOP::GFP UPR reporter cell lines with plasmids UK1397, UK1398 or UK1443

637 (see Supplementary File 1) using Lipofectamine LTX. Where indicated, cells were treated 24

638 hours after transfection with the ER stress-inducing agent tunicamycin (2.5 μg/µl) for 16

639 hours to activate CHOP::GFP (which results in enhanced GFP production). Cells were

640 analyzed by dual-channel flow cytometry with an LSRFortessa cell analyzer (BD

641 Biosciences) as described previously (Tsunoda et al., 2014). GFP (excitation laser 488 nm,

642 filter 530/30) and mCherry signals (excitation laser 561, filter 610/20) were detected. The

643 data were processed using FlowJo and median reporter values were plotted using

644 GraphPad Prism (GraphPad Software).

645 Mammalian cell lysates

646 Cell lysis was performed as described (Preissler et al., 2015). Mammalian cells were grown

647 on 10 cm dishes and treated as indicated. At the end of each experiment the dishes were

648 placed on ice and washed twice with ice-cold PBS. The cells were detached with a cell

649 scraper in PBS containing 1 mM EDTA, transferred to a 1.5 ml reaction tube and centrifuged

650 for 5 minutes at 370 g at 4°C. The cells were lysed in HG lysis buffer [20 mM HEPES-KOH

651 pH 7.4, 150 NaCl, 2 mM MgCl2, 10 mM D-glucose, 10% (v/v) glycerol, 1% (v/v) Triton X-100]

652 supplemented with protease inhibitiors (1 mM PMSF, 2 µg/ml pepstatin, 2 µg/ml leupeptin, 4

653 µg/ml aprotinin) for 10 minutes on ice and centrifuged at 21,000 g for 10 minutes at 4°C. The

26 BiP AMPylation

654 protein concentrations of the cleared lysates were determined using the BIO-RAD protein

655 assay reagent (BioRad) and normalized with lysis buffer (usually the protein concentration

656 was adjusted to 1.2 µg/µl). Where indicated the lysates were treated with SubA protease as

657 described. For SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the proteins

658 were denatured by addition of SDS sample buffer and heating for 10 minutes at 75°C

659 followed by separation on 12% SDS polyacrylamide gels.

660 For detection of endogenous BiP by native-PAGE (see below) the cells were lysed in HG

661 lysis buffer containing 2 x protease inhibitors (2 mM PMSF, 4 µg/ml pepstatin, 4 µg/ml

662 leupeptin, 8 µg/ml aprotinin) with or without 100 U/ml hexokinase (Type III from baker’s

663 yeast; Sigma) as described above and samples were loaded immediately on native or SDS

664 polyacrylamide gels. Phosphatase inhibitors (10 mM tetrasodium pyrophosphate, 100 mM

665 sodium fluoride, 17.5 mM β-glycerophosphate) were added to the lysis buffer in experiments

666 where phosphorylated proteins were detected.

667 Native polyacrylamide gel electrophoresis (native-PAGE)

668 Native-PAGE was performed as described (Preissler et al., 2015). Tris-glycine

669 polyacrylamide gels consisting of a 4.5% stacking gel and a 7.5% separation gel were used

670 to separate purified protein or proteins from mammalian cell lysates under non-denaturing

671 conditions to detect BiP oligomers. The gels were run in a Mini-PROTEAN electrophoresis

672 chamber (BioRad) in running buffer (25 mM Tris, 192 mM glycine, pH ~8.8) at 120 V for 2

673 hours when cell lysates were applied or for 1:45 hours when His6-tagged purified BiP

674 proteins were analyzed. The proteins were then visualized by staining with InstantBlue

675 Coomassie solution (expedeon) or transferred to a polyvinylidene difluoride (PVDF)

676 membrane in blotting buffer (48 mM Tris, 39 mM glycine, pH ~9.2) containing 0.04 (w/v)

677 SDS for 16 hours at 30 V for immunodetection. The membrane was washed after the

678 transfer for 20 minutes in blotting buffer supplemented with 20% (v/v) methanol before

679 blocking. Seven µg of purified BiP protein was loaded per lane on a native gel to detect BiP

27 BiP AMPylation

680 oligomers by Coomassie staining and volumes of lysates corresponding to 30 µg of protein

681 (CHO-K1 and AR42j cells) or 90 µg protein (Flp-In T-REx 293 cells) were loaded per lane.

682 Immunoblot analysis

683 Proteins were separated by SDS-PAGE or native-PAGE and transferred to PVDF

684 membranes. The membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25

685 mM Tris-HCl pH 7.5, 150 mM NaCl) and probed with primary antibodies followed by IRDye

686 fluorescently labeled secondary antibodies (LiCor). The membranes were scanned with an

687 Odyssey near-infrared imager (LiCor) and where indicated densitometric quantification of the

688 immunoblot signals was performed with ImageJ (NIH). Primary antibodies and antisera

689 against hamster BiP [chicken anti-BiP; (Avezov et al., 2013)], eIF2α [mouse anti-eIF2α;

690 (Scorsone et al., 1987)], phosphorylated eIF2α [rabbit anti-eIF2α Phospho (pS51); Epitomics

691 cat. # 1090-1], and FICD [rabbit anti-FICD; LifeSpan BioSciences cat. # LS-C80941, or

692 chicken anti-FICD (see below)] were used.

693 Phos-tag gel electrophoresis

694 To separate unmodified IRE1α and phosphorylated IRE1α lysates from AR42j cells were

695 loaded on SDS polyacrylamaide gels (Mini-PROTEAN system; Bio-Rad) consisting of a 4%

696 polyacrylamide stacking gel and a 7% polyacrylamide separation gel. The separation gel

697 contained 50 µM MnCl2 and 25 µM Phos-tag Acrylamide AAL-107 (NARD Institute Ltd.,

698 Amagasaki, Japan). The gels were run for 3 hours at 300 V, incubated for 10 minutes in

699 immunoblot transfer buffer containing 1 mM EDTA and proteins were transferred to PVDF

700 membranes.

701 Protein purification

702 Wildtype and mutant versions of N-terminally hexahistidine- (His6-) tagged hamster BiP

703 proteins (see Supplementary File 1 and “Plasmid construction”) were expressed in M15 E.

704 coli cells (Qiagen) as described (Preissler et al., 2015). Bacterial cultures were grown at

705 37°C in LB medium containing 100 µg/ml ampicillin and 50 µg/ml kanamycin to an optical

706 density (OD600 nm) of 0.8 and expression was induced with 1 mM isopropylthio β-D-1-

28 BiP AMPylation

707 galactopyranoside (IPTG). After incubation for 6 hours at 37 ˚C the cells were harvested by

708 centrifugation and pellets were lysed with a high-pressure homogenizer (EmulsiFlex-C3,

709 Avestin) in buffer A [50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM MgCl2, 0.2% (v/v) Triton

710 X-100, 10% (v/v) glycerol, 20 mM imidazole] containing protease inhibitors [2 mM

711 phenylmethylsulphonyl fluoride (PMSF), 4 µg/ml pepstatin, 4 µg/ml leupeptin, 8 µg/ml

712 aprotinin] and 0.1 mg/ml DNaseI. The lysates were cleared by centrifugation (30 minutes at

713 25,000 g) and incubated with 1 ml nickel affinity matrix (Ni-NTA agarose; Quiagen) per 1 l of

714 expression culture for 2 hours at 4°C. The beads were washed five times with 20 bed

715 volumes of buffer A sequentially supplemented with (i) 30 mM imidazole, (ii) 1% (v/v) Triton

716 X-100, (iii) 1 M NaCl, (iv) 5 mM Mg2+-ATP, or (v) 0.5 M Tris-HCl pH 7.5. Bound BiP proteins

717 were eluted in buffer B [50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM MgCl2, 10% (v/v)

718 glycerol, 250 mM imidazole] and dialyzed against HKM buffer [50 mM HEPES-KOH pH 7.4,

719 150 mM KCl, 10 mM MgCl2]. The purified proteins were concentrated using centrifugal filters

720 (Amicon Ultra, 30 kDa MWCO; Merck Millipore), snap-frozen in liquid nitrogen and stored at

721 -80°C. The purification of mature hamster BiP (19-654) was described in detail in (Preissler

722 et al., 2015).

723 Mammalian expressed GST-tagged recombinant FICDE234G was produced in HEK293T cells.

724 The cells were grown on 10 cm tissue culture dishes to a density of 80% and transfected

725 either with plasmid encoding only GST or GST-FICDE234G (UK1415) using the

726 polyethylenimine (PEI) method with the following adaptations: 14 µg plasmid DNA was

727 added to 700 µl Opti-MEM (Gibco) mixed with 56 µl of PEI (1 mg/ml stock) and added

728 dropwise to the cells after incubation for 20 minutes at room temperature (ten dishes were

729 usually transfected per preparation). 36 hours after transfection the cells were lysed as

730 described above with Triton lysis buffer [20 mM HEPES-KOH pH 7.4, 150 NaCl, 1 mM

731 EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100] containing protease inhibitors. The

732 combined lysate (from ten dishes) was incubated with 200 µl GSH-Sepharose beads

733 (Glutathione Sepharose 4B; GE Healthcare) for 3 hours at 4°C. The beads were recovered

29 BiP AMPylation

734 by centrifugation for 5 minutes at 500 g, washed twice with Triton lysis buffer containing 300

735 mM NaCl and twice with AMPylation buffer [25 mM HEPES-KOH pH 7.4, 100 mM KCl, 4

736 mM MgCl2, 1 mM CaCl2, 0.1% (v/v) Triton X-100]. The beads with bound protein were stored

737 in AMPylation buffer containing 50% (v/v) glycerol at -20°C and used for radioactive in vitro

738 AMPylation reactions.

739 For bacterial expression of N-terminally GST-tagged FICD proteins and ERdj6 J-domains,

740 plasmid DNA encoding active GST-FICDE234G (UK1479), inactive GST-FICDE234G-H363A

741 (UK1480), GST-J (UK185) or mutant GST-J* (UK186) was transformed into BL21 T7

742 Express lysY/Iq E. coli cells (New England BioLabs cat. # C3013). Bacterial cultures were

743 grown at 37°C in LB medium containing 100 µg/ml ampicillin to OD600 nm 0.8, shifted to 20°C

744 and expression was induced with 0.1 mM IPTG for 16 hours. Afterwards, the cells were

745 harvested and lysed as described above in lysis buffer [50 mM Tris-HCl pH 7.5, 500 mM

746 NaCl, 1 mM MgCl2, 2 mM dithiothreitol (DTT), 0.2% (v/v) Triton X-100, 10% (v/v) glycerol]

747 containing protease inhibitors and DNaseI. The lysates were cleared by centrifugation (30

748 minutes at 25,000 g) and incubated with 0.7 ml glutathione-Sepharose beads per 1 l of

749 expression culture for 3 hours at 4°C. The beads were transferred to a column and washed

750 with 20 ml wash buffer B [50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM DTT, 0.2% (v/v)

751 Triton X-100, 10% (v/v) glycerol] containing protease inhibitors, 20 ml wash buffer C [50 mM

752 Tris-HCl pH 7.5, 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1% (v/v) Triton X-100, 10%

753 (v/v) glycerol] containing protease inhibitors and 20 ml wash buffer C sequentially

754 supplemented (i) 1% (v/v) Triton X-100, (ii) 1 M NaCl, (iii) 3 mM ATP, (iv) or 0.5 M Tris-HCl

755 pH 7.5. Bound BiP proteins were then eluted in elution buffer [50 mM HEPES-KOH pH 7.4,

756 100 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, 40 mM

757 reduced glutathione] and concentrated protein solutions were frozen in liquid nitrogen and

758 stored at -80°C.

759 N-terminally His6-Smt3 tagged wildtype mouse FICD lumenal domain (L104-P458)

760 (UK1564) was encoded on a pET28b plasmid (Novagen) and expressed in E. coli BL21 T7

30 BiP AMPylation

761 Express lysY/Iq cells. Bacterial cultures (3 liters) were grown at 37°C in LB medium

762 containing 100 µg/ml ampicillin to OD600 nm 0.8 and expression was induced with 0.1 mM

763 IPTG. After incubation for 16 hours at 20°C the cells were sedimented by centrifugation and

764 pellets were lysed as described above in lysis buffer [50 mM Tris-HCl pH 7.4, 500 mM NaCl,

765 1 mM MgCl2, 10% (v/v) glycerol, 30 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine

766 (TCEP)] supplemented with protease inhibitors and DNaseI. The lysates were cleared by

767 centrifugation for 30 minutes at 33,000 g, followed by addition of 0.2% (v/v) Triton X-100 and

768 0.002% (v/v) sodium deoxycholate, and incubated with 1 ml Ni-NTA agarose (Quiagen) for

769 2.5 hours at 4°C. The beads were washed with 25 bed volumes of wash buffer [50 mM Tris-

770 HCl pH 7.4, 500 mM NaCl, 10% (v/v) glycerol, 0.2% (v/v) Triton X-100, 1 mM TCEP,

771 protease inhibitors], once with buffer D [50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% (v/v)

772 glycerol, 0.1% (v/v) Triton X-100, 10 mM MgCl2, 1 mM TCEP, and protease inhibitors], and

773 then with buffers Ei-iv [buffer D supplemented with (i) 1% (v/v) Triton X-100, (ii) 1 M NaCl, (iii)

774 3 mM ATP, or (iv) 0.5 M Tris-HCl pH 7.4]. Bound mFICD protein was eluted in buffer F [50

775 mM Tris-HCl pH 7.4, 100 mM NaCl, 4 mM MgCl2, 1 mM CaCl2, 10% (v/v) glycerol, 250 mM

776 imidazole, 1 mM TCEP), and dialyzed against 50 mM HEPES-NaOH pH 7.4, 100 mM NaCl,

777 0.5 mM 2-mercaptoethanol in the presence of Ulp1 (2 µg per mg of eluted protein) for 16

778 hours at 4°C to cleave the His6-Smt3 tag. The dialyzed and cleaved protein was then diluted

779 1:2 in 25 mM HEPES-NaOH pH 8.1 to reduce the ionic strength, loaded on a anion-

780 exchange chromatography column (Mono Q 5/50 GL; GE Healthcare) and eluted with a

781 linear salt gradient from 50 to 500 mM NaCl in 25 mM HEPES-NaOH pH 7.4. The mFICD-

782 containing fractions were pooled and further purified by size-exclusion chromatography

783 (Superdex 200 10/300; GE Healthcare) in gel filtration buffer (25 mM HEPES-NaOH pH 7.4,

784 150 mM NaCl). The eluted fractions were combined and provided to Aves Labs (Oregon,

785 USA) as an immunogen for production of chicken anti-FICD antibodies.

31 BiP AMPylation

786 Immunoaffinity purification and immunoblotting of FICD

787 The absence of FICD protein in FICD-/- AR42j cells was confirmed by immunoaffinity

788 purification as the endogenous protein could not be detected by immunoblotting total lysates.

789 Protein extracts from wildtype and FICD-/- AR42j cells were prepared in HG lysis buffer

790 containing protease inhibitors, cleared, normalized and equal volumes of the lysates (4 mg

791 total protein) were incubated with 15 µl UltraLink Hydrazine Resin (Pierce cat. # 53149) on

792 which FICD-specific chicken IgY antibodies have been covalently immobilized according to

793 the manufacturer’s instructions (chicken anti-FICD) or rabbit polyclonal IgG antibodies

794 against human FICD (rabbit anti-FICD; LifeSpan BioSciences) bound to Protein A

795 Sepharose 4B beads (Invitrogen cat. #10-1041), for 16 hours at 4°C. The beads were then

796 recovered by centrifugation for 1 minute at 1,000 g and washed three times with HG lysis

797 buffer. Bound proteins were eluted in 40 µl 2 x SDS sample buffer for 10 minutes at 70°C

798 and equal volumes of the samples were loaded on a 12% SDS polyacrylamide gel, and

799 endogenous FICD was detected by immunoblotting with rabbit anti-FICD or chicken anti-

800 FICD antibodies. Samples of the normalized lysates (25 µg) were loaded and BiP was

801 detected as an “input” control.

802 In vitro AMPylation assay

803 Unless indicated otherwise, in vitro AMPylation was performed in AMPylation buffer

804 (described above) by incubation of purified wildtype or mutant BiP proteins at 20 µM with

805 bacterially expressed GST-FICDE234G (or as a control with inactive GST-FICDE234G-H363A) at

806 0.8 µM in presence of 1.5 mM ATP for 45 minutes at 30°C. The final volume of a typical

807 reaction was 15 µl. The samples were then treated with or without the BiP linker-specific

808 protease SubA at 30 ng/µl for 10 minutes at room temperature. Volumes corresponding to 7

809 µg BiP protein were loaded per lane on native or SDS gels and visualized by Coomassie

810 staining. The in vitro AMPylation time-course experiments presented in Figure 7C and D

811 were performed likewise but after incubation for the indicated times at 30°C the reactions

32 BiP AMPylation

812 were stopped on ice by addition of 40 mM EDTA and samples were run immediately on

813 native gels.

814 Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or

815 mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32P-ATP (EasyTide;

816 Perkin Elmer) in a total volume of 30 µl in AMPylation buffer. The reactions were incubated

817 with 5 µl glutathione-sepharose beads coupled with mammalian expressed GST-FICDE234G

818 (or GST only) in 1.5 ml reaction tubes at 30°C on a thermomixer (Eppendorf) while shaking

819 at 800 rpm. After 45 minutes [in these experiments a time before the reaction went to

820 completion to reveal differences in AMPylation amongst BiP mutants (Figure 5A and C and

821 Figure 7A)] the beads were sedimented by centrifugation and the reaction was terminated by

822 addition of SDS loading buffer to the supernatants and heating for 5 minutes at 75°C. Equal

823 volumes of the samples were loaded on 12% SDS polyacrylamide gels. Proteins were

824 visualized by Coomassie staining and radioactive signals were detected with a Typhoon Trio

825 imager (GE Healthcare) upon overnight exposure of the dried gels to a storage phosphor

826 screen. To test the sensitivity of AMPylated BiP towards cleavage by SubA, 6 µg of purified

827 BiPT229A were labeled in 60 µl AMPylation buffer containing 6.7 MBq of α-32P-ATP and 10 µl

828 of GST-FICDE234G-coupled beads as described above. After incubation for 30 minutes at

829 30°C non-radioactive ATP was added at 5 µM and the reaction was allowed to proceed for

830 another 60 minutes. The beads were then sedimented by centrifugation and 1.5 mM ATP

831 was added to the supernatant. To remove nucleotides the sample was passed through a

832 Sephadex G-50 MicroSpin column (illustra AutoSeq G-50; GE Healthcare) equilibrated with

833 AMPylation buffer. 1.5 µg of the recovered protein was mixed with 60 µg unmodified BiPT229A

834 (at 0.6 mg/ml final) in presence of 1.5 mM ATP and the combined sample was divided into

835 fractions to which SubA (from a dilution series) was added to yield final concentrations

836 between 0.08 and 120 ng/µl. After incubation for 30 minutes at room temperature the

837 proteins were denatured with SDS sample buffer and equal volumes were applied to SDS-

838 PAGE, Coomassie-stained and signals were detected by autoradiography.

33 BiP AMPylation

839 Purification of in vitro AMPylated BiP proteins

840 Twenty mg of purified wiltype BiP or BiPV461F protein were in vitro AMPylated for 2 hours at

841 30°C with 1 mg bacterially expressed GST-FICDE234G in presence of 1.5 mM ATP in

842 AMPylation buffer in a final volume of 12 ml. BiP proteins were then bound to 400 µl nickel

843 affinity matrix, washed with AMPylation buffer and eluted in AMPylation buffer containing

844 250 mM imidazole. The concentrated eluate was divided into aliquots and immediately

845 frozen in liquid nitrogen and stored at -80°C. For applications that required complete

846 absence of detergent or imidazole the affinity chromatography eluate was passed through a

847 CentriPure P25 desalting column (emp BIOTECH) equilibrated in HKM buffer followed by

848 concentration of the proteins using centrifugal filters (Amicon Ultra, 30 kDa MWCO; Merck

849 Millipore) before freezing. The in vitro modified proteins were used for mass spectrometry

850 analyses and functional assays (see below). Unmodified BiP from parallel mock in vitro

851 AMPylation reactions, to which no enzyme has been added, served as controls in all assays.

852 Preparation of SILAC samples

853 The experimental strategy for the stable isotope labeling by amino acids in cell culture

854 (SILAC) experiment is outlined in Figure 6A and samples were prepared as follows: Wildtype

855 and FICD-/- CHO-K1 cells were adapted to Ham’s F12 medium minus L-arginine and L-

856 for SILAC (Pierce cat. # 88424) supplemented with 10% (v/v) dialyzed fetal bovine serum

857 (Gibco, Life Technologies cat. # 26400-044), 1 x Penicillin-Streptomycin (Sigma), 2 mM L-

858 glutamine (Sigma), 280 mg/l L-proline (Sigma cat. # P5607-25G), 62.5 mg/l “light” L-lysine

859 monohydrochloride (Sigma cat. # L8662-25G) and 60.5 mg/l “light” L-arginine

860 monohydrochloride (Sigma cat. # A6969-25G) and incubated as described above. Once

861 adapted, the cells were cultured in SILAC medium containing either 60.5 mg/l “light” (as

862 above) or “heavy” L-arginine monohydrochloride (R10; U-13C6, U-15N4; CK Gas Products

863 cat. # CNLM-539) for several passages (> 15 cell divisions) before expansion in five 10 cm

864 dishes per sample. The cells were grown to ~70% confluence and medium was exchanged

865 14 hours before treatment with or without 100 µg/ml cycloheximide for 3 hours. After

34 BiP AMPylation

866 washing with ice-cold PBS the cells were collected in PBS containing 1 mM EDTA and lysed

867 in Triton lysis buffer [20 mM HEPES-KOH pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v)

868 Triton X-100, 10% (v/v) glycerol] containing protease inhibitors. The protein concentrations

869 of the cleared lysates were normalized and equal volumes were then mixed (1:1 ratio) as

870 indicated. Each combined sample was pre-cleared for 15 minutes at 4°C with 30 µl blocked

871 UltraLink Hydrazine Resin followed by immunoaffinity purification for 2 hours at 4°C with 30

872 µl of the same resin to which BiP-specific IgY antibodies were covalently coupled (Preissler

873 et al., 2015). The beads were washed twice for 10 minutes with lysis buffer, once with lysis

874 buffer containing 350 mM NaCl and once again with lysis buffer, before elution in 2x non-

875 reducing SDS sample buffer for 10 minutes at 70°C. Afterwards, the beads were removed by

876 centrifugation and the supernatants were supplemented with 125 mM DTT, heated for 5

877 minutes at 70°C and loaded on a 12% SDS polyacrylamide gel. The gel was stained with

878 Coomassie and bands corresponding in size to BiP were cut out for in-gel digest with Arg-C

879 endopeptidase, and analysis by mass spectrometry.

880

881 Immunoaffinity purification of intact endogenous BiP for mass determination

882 Wildtype and FICD-/- CHO-K1 cells were grown in standard medium (ten 10 cm dishes per

883 sample) and treated for 3 hours with 100 µg/ml cycloheximide before cell lysis with Triton

884 lysis buffer supplemented with protease inhibitors and BiP was immunoaffinity purified as

885 described above with the following modifications: The lysates (~4 mg protein per sample)

886 were pre-cleared with 40 µl blocked UltraLink Hydrazine Resin for 30 minutes at 4°C

887 followed by incubation for 2 hours with 60 µl of the resin carrying covalently attached BiP-

888 specific IgY antibodies. The beads were washed sequentially for 10 minutes with lysis buffer,

889 lysis buffer containing 350 mM NaCl, HG buffer (see above) containing 1 mM ATP, and once

890 again with lysis buffer. Bound protein was eluted in 150 µl of 0.2 M glycine-HCl pH 2.5 for 10

891 minutes at 16°C followed by neutralization of the recovered sample supernatants with 1/10

35 BiP AMPylation

892 volume of 1.5 M Tris-HCl pH 8.8. Afterwards, protein was precipitated with isopropanol and

893 analyzed by mass spectrometry.

894

895 Mass spectrometry

896 Molecular masses of intact BiP proteins were measured by electrospray ionization mass

897 spectrometry using a Q-Trap 4000 (ABSciex) after ‘on-line’ reverse-phase chromatographic

898 purification of samples as described previously (Carroll et al., 2009). The instrument was

899 operated in MS mode, and was calibrated with a mixture of myoglobin and trypsinogen.

900 Reconstructed molecular masses were calculated with Bioanalyst (ABSciex) and masses

901 from amino acid sequences with MassLynx (Waters).

902 To analyze masses of BiP peptides, proteins were reduced, alkylated and digested “in-gel”.

903 In vitro AMPylated BiP protein was digested with the proteolytic enzymes as indicated and

904 SILAC samples were digested using Arg-C. The resulting peptides were analysed by LC-

905 MS/MS using either an Orbitrap XL coupled to a nanoAcquity UHPLC or a Q Exactive

906 coupled to a RSLC3000 UHPLC. Data was processed in Proteome Discoverer 1.4 using

907 Sequest to search a Uniprot E. coli database (downloaded 29/04/15, 4,378 entries) with the

908 sequence for Chinese hamster (Cricetulus griseus) BiP added or a Chinese Hamster

909 database (downloaded 19/02/14, 23,884 entries). Oxidation (M), (N/Q) and

910 AMPylation (S/T) were set as variable modifications and carbamidomethylation (C) as a

911 fixed modification. FDR calculations were performed by Percolator and peptides were

912 filtered to 1%. Peak areas were determined using the Precursor Ion Quantifier node in

913 Proteome Discoverer.

914 Isoelectric focusing (IEF)

915 Purified untagged hamster BiP19-654 at 15 µM was in vitro AMPylated with 0.75 µM GST-

916 FICDE234G (or as a control with inactive GST-FICDE234G-H363A) in presence of 1.5 mM ATP for

917 the indicated times at 30°C as described above. The reactions were then treated without or

918 with 30 ng/µl SubA for 10 minutes at room temperature in a final volume of 20 µl and

36 BiP AMPylation

919 analyzed by IEF as described previously (Chambers et al., 2012; Laitusis et al., 1999) with

920 modifications: The samples were diluted with 180 µl of IEF sample buffer [8 M urea, 5%

921 (w/v) CHAPS, 50 mM DTT, 2% (v/v) Pharmalyte (pH 4.5-5.4; GE Healthcare)] and loaded on

922 a 3.75% polyacrylamide gel containing 8.8 M urea, 1.25% (w/v) CHAPS and 5% (v/v)

923 Pharmalyte. The loaded samples were overlaid with 0.5 M urea and 2% (v/v) Pharmalyte

924 solution before separation. 10 mM glutamic acid and 50 mM histidine were used as anode

925 and cathode buffers, respectively. After the run (100V, 10 minutes; 250V, 1 hour; 300V, 1

926 hour; 500V, 30 minutes) proteins were transferred to a nitrocellulose membrane in blotting

927 buffer [25 mM Tris-HCl pH 9.2, 190 mM glycine, 0.01% (w/v) SDS, 10% (v/v) methanol] for 3

928 hours at 300 mA and immunodetected with BiP-specific antibodies as described above.

929 Lysate samples from mammalian cells (CHO-K1 and AR42j) were prepared for analysis by

930 IEF as follows: Cells were grown in 10 cm dishes to approximately 90% confluence, washed

931 with ice-cold TBS, resuspended in 1 ml TBS, sedimented by centrifugation, and lysed in 30 x

932 its packed cell volume of IEF lysis buffer [8.8 M urea, 5% (w/v) CHAPS, 1 µM sodium

933 pyrophosphate, 2 mM imidodiphosphate, 50 mM DTT and 2% (v/v) Pharmalyte] at room

934 temperature for 5 minutes. The lysates were then centrifuged at 20,238 g for 10 minutes at

935 room temperature, the supernatants transferred to a new tube and then centrifuged again for

936 60 minutes. The cleared lysates (50 µl) were then purified using Sephadex G-50 MicroSpin

937 columns equilibrated with IEF sample buffer, diluted 1:2 in IEF sample buffer and 15 µl

938 loaded on a 3.75% polyacrylamide gel followed by BiP immunoblotting as described above.

939 Malachite green ATPase assay

940 Malachite green (MG) reaction solution was prepared freshly by mixing stock solutions of

941 MG dye [0.111% (w/v) malachite green, 6 N sulphuric acid], 7.5% (w/v) ammonium

942 molybdate and 11% (v/v) Tween 20 in a 10:2.5:0.2 ratio. Samples of purified BiP proteins (5

943 µM) were incubated in HKM with 3 mM ATP in a final volume of 20 µl for 60 minutes at 30°C.

944 15 µl of each sample were then diluted with 135 µl water on a 96-well plate, mixed with 50 µl

945 MG reaction solution and incubated for 2 minutes at room temperature. 20 µl of a 34% (w/v)

37 BiP AMPylation

946 sodium citrate solution were added to quench the reactions and after incubation for further

947 30 minutes the absorbance was measured at 612 nm with a plate reader (Infinite F500;

948 Tecan). Standard curves were prepared with serial dilutions of KH2PO4 and served as a

949 reference to calculate Vmax values. Statistical analysis was by unpaired t-test performed

950 using Graphpad Prism version 6.

951 Fluorescence polarization (FP) measurements

952 Binding of substrate peptide by purified BiP proteins was measured as previously described

953 (Chambers et al., 2012; Marcinowski et al., 2011) with modifications: Unmodified or in vitro

954 AMPylated BiP proteins were incubated at the indicated concentrations (between 0 and 40

955 µM) with 1 µM lucifer yellow (LY)-labeled substrate peptide (HTFPAVLGSC) in presence of

956 1 mM ADP for 24 hours at 30°C in FP buffer [37.5 mM HEPES-KOH pH 7.4, 125 mM KCl, 7

957 mM MgCl2, 0.5 mM CaCl2, 125 mM imidazole, 0.05% (v/v) Triton X-100]. 20 µl of each

958 sample were transferred to a 386-well polystyrene microplate (µClear, black; greiner bio-one

959 cat. # 781096) and fluorescence polarization of the lucifer yellow fluorophore was measured

960 (excitation λ = 430 nm; emission λ = 535 nm) at room temperature in a plate reader. For

961 analysis of competitive substrate dissociation, BiP proteins (40 µM) were incubated with LY-

962 labeled substrate peptide (1 µM) as described above. Afterwards, 400-fold molar excess of

963 unlabeled substrate peptide (HTFPAVL) was added to the pre-formed BiP:substrate

964 complexes and the change in fluorescence polarization was measured over time. From each

965 value the background signal of a reference samples containing only LY-labeled peptide

966 (without BiP protein) were subtracted.

967 Reverse transcription of cellular mRNA and quantitative PCR of mRNA abundance

968 RNA from CHO-K1 cells was extracted using RNA Stat-60 (Amsbio) according to the

969 manufacturer’s instructions. 1 µg RNA was reverse transcribed with Oligo(dT)18 primer

970 using RevertAid Reverse Transcriptase (Thermo Scientific). Quantitative PCR analysis was

971 performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) according

972 to the manufacturer’s instructions on a 7900HT Fast Real-Time PCR System (Applied

38 BiP AMPylation

973 Biosystems). FICD, RPL27 (60S ribosomal protein L27) and PPIA (cyclophilin A) were

974 amplified using the following primers: FICD-F 5’-GGGCTGCTGACTGTTAAGACTA-3’,

975 FICD-R 5’-CCTTGACGCTTCATCTCCA-3’, RPL27-F 5’-ACAATCACCTCATGCCCACAAG-

976 3’, RPL27-R 5’-GCGTTTCAGGGCTGGGTCTC-3’, PPIA-F 5’-

977 TTCCTCCTTTCACAGAATTATCCC-3’, and PPIA-R 5’-CTGCCGCCAGTGCCATTATG-3’.

978 Relative quantities of amplified cDNAs were then determined using SDS 2.4.1 software

979 (Applied Biosystems) and normalized to PPIA mRNA.

980

981

982

983

39 BiP AMPylation

984 Acknowledgments

985 We thank the CIMR flow cytometry core facility team (Reiner Schulte, Michal Maj and Chiara

986 Cossetti) for assistance with FACS and Heather P. Harding, Niko Amin-Wetzel and Joseph

987 E. Chambers (all from CIMR, Cambridge) for technical advice and critical input and Qinlian

988 Liu from Virginia Commonwealth University for sharing unpublished structural data.

989 Supported by grants from the Wellcome Trust (Wellcome 084812/Z/08/Z) the European

990 Commission (EU FP7 Beta-Bat No: 277713), a Wellcome Trust Strategic Award for core

991 facilities to the Cambridge Institute for Medical Research (Wellcome 100140). DR is a

992 Wellcome Trust Principal Research Fellow.

993

994 Competing Interests

995 The authors have no financial or non-financial competing interests.

996

40 BiP AMPylation

997 References

998 Avezov, E., Cross, B.C., Kaminski Schierle, G.S., Winters, M., Harding, H.P., Melo, E.P., 999 Kaminski, C.F., and Ron, D. (2013). Lifetime imaging of a fluorescent protein sensor reveals 1000 surprising stability of ER thiol redox. J Cell Biol 201, 337-349.

1001 Balch, W.E., Morimoto, R.I., Dillin, A., and Kelly, J.W. (2008). Adapting proteostasis for 1002 disease intervention. Science 319, 916-919. 1003 Bertolotti, A., Zhang, Y., Hendershot, L., Harding, H., and Ron, D. (2000). Dynamic 1004 interaction of BiP and the ER stress transducers in the unfolded protein response. Nat Cell 1005 Biol 2, 326-332. 1006 Broncel, M., Serwa, R.A., Bunney, T.D., Katan, M., and Tate, E.W. (2015). Global profiling of 1007 HYPE mediated AMPylation through a chemical proteomic approach. Mol Cell Proteomics.

1008 Bunney, T.D., Cole, A.R., Broncel, M., Esposito, D., Tate, E.W., and Katan, M. (2014). 1009 Crystal structure of the human, FIC-domain containing protein HYPE and implications for its 1010 functions. Structure 22, 1831-1843.

1011 Carlsson, L., and Lazarides, E. (1983). ADP-ribosylation of the Mr 83,000 stress-inducible 1012 and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and 1013 glucose starvation. Proc Natl Acad Sci U S A 80, 4664-4668.

1014 Carroll, J., Fearnley, I.M., Wang, Q., and Walker, J.E. (2009). Measurement of the molecular 1015 masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single 1016 experiment. Anal Biochem 395, 249-255. 1017 Chambers, J.E., Petrova, K., Tomba, G., Vendruscolo, M., and Ron, D. (2012). ADP 1018 ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein 1019 load. J Cell Biol 198, 371-385. 1020 Chang, S.C., Erwin, A.E., and Lee, A.S. (1989). Glucose-regulated protein (GRP94 and 1021 GRP78) genes share common regulatory domains and are coordinately regulated by 1022 common trans-acting factors. Mol Cell Biol 9, 2153-2162.

1023 De Los Rios, P., and Barducci, A. (2014). Hsp70 chaperones are non-equilibrium machines 1024 that achieve ultra-affinity by energy consumption. Elife 3, e02218. 1025 Dorner, A., Wasley, L., and Kaufman, R. (1992). Overexpression of GRP78 mitigates stress 1026 induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese 1027 hamster ovary cells. EMBO J 11, 1563-1571.

1028 Engel, P., Goepfert, A., Stanger, F.V., Harms, A., Schmidt, A., Schirmer, T., and Dehio, C. 1029 (2012). Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins. 1030 Nature 482, 107-110.

41 BiP AMPylation

1031 Fernandez-Saiz, V., Moro, F., Arizmendi, J.M., Acebron, S.P., and Muga, A. (2006). Ionic 1032 contacts at DnaK substrate binding domain involved in the allosteric regulation of lid 1033 dynamics. J Biol Chem 281, 7479-7488.

1034 Freiden, P.J., Gaut, J.R., and Hendershot, L.M. (1992). Interconversion of three differentially 1035 modified and assembled forms of BiP. EMBO J 11, 63-70. 1036 Garcia-Pino, A., Zenkin, N., and Loris, R. (2014). The many faces of Fic: structural and 1037 functional aspects of Fic enzymes. Trends Biochem Sci 39, 121-129.

1038 Gaut, J.R. (1997). In vivo threonine phosphorylation of immunoglobulin binding protein (BiP) 1039 maps to its protein binding domain. Cell Stress Chaperones 2, 252-262.

1040 Gaut, J.R., and Hendershot, L.M. (1993). Mutations within the nucleotide binding site of 1041 immunoglobulin-binding protein inhibit ATPase activity and interfere with release of 1042 immunoglobulin heavy chain. J Biol Chem 268, 7248-7255.

1043 Ham, H., Woolery, A.R., Tracy, C., Stenesen, D., Kramer, H., and Orth, K. (2014). Unfolded 1044 protein response-regulated Drosophila Fic (dFic) protein reversibly AMPylates BiP 1045 chaperone during endoplasmic reticulum homeostasis. J Biol Chem 289, 36059-36069. 1046 Hendershot, L.M., Ting, J., and Lee, A.S. (1988). Identity of the immunoglobulin heavy- 1047 chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of 1048 posttranslational modifications in its binding function. Mol Cell Biol 8, 4250-4256. 1049 Kinch, L.N., Yarbrough, M.L., Orth, K., and Grishin, N.V. (2009). Fido, a novel AMPylation 1050 domain common to fic, doc, and AvrB. PLoS One 4, e5818. 1051 Kityk, R., Kopp, J., Sinning, I., and Mayer, M.P. (2012). Structure and dynamics of the ATP- 1052 bound open conformation of Hsp70 chaperones. Mol Cell 48, 863-874.

1053 Klampfl, T., Gisslinger, H., Harutyunyan, A.S., Nivarthi, H., Rumi, E., Milosevic, J.D., Them, 1054 N.C., Berg, T., Gisslinger, B., Pietra, D., et al. (2013). Somatic mutations of calreticulin in 1055 myeloproliferative neoplasms. N Engl J Med 369, 2379-2390.

1056 Kozutsumi, Y., Segal, M., Normington, K., Gething, M.J., and Sambrook, J. (1988). The 1057 presence of malfolded proteins in the endoplasmic reticulum signals the induction of 1058 glucose-regulated proteins. Nature 332, 462-464. 1059 Laitusis, A.L., Brostrom, M.A., and Brostrom, C.O. (1999). The dynamic role of GRP78/BiP 1060 in the coordination of mRNA translation with protein processing. J Biol Chem 274, 486-493.

1061 Laufen, T., Mayer, M.P., Beisel, C., Klostermeier, D., Mogk, A., Reinstein, J., and Bukau, B. 1062 (1999). Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl 1063 Acad Sci U S A 96, 5452-5457. 1064 Ledford, B.E., and Jacobs, D.F. (1986). Translational control of ADP-ribosylation in 1065 eucaryotic cells. Eur J Biochem 161, 661-667.

42 BiP AMPylation

1066 Leno, G.H., and Ledford, B.E. (1989). ADP-ribosylation of the 78-kDa glucose-regulated 1067 protein during nutritional stress. Eur J Biochem 186, 205-211.

1068 Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991). Escherichia coli 1069 DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl 1070 Acad Sci U S A 88, 2874-2878. 1071 Logsdon, C.D., Moessner, J., Williams, J.A., and Goldfine, I.D. (1985). Glucocorticoids 1072 increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar 1073 AR42J cells. J Cell Biol 100, 1200-1208. 1074 Marcinowski, M., Holler, M., Feige, M.J., Baerend, D., Lamb, D.C., and Buchner, J. (2011). 1075 Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated 1076 conformational transitions. Nat Struct Mol Biol 18, 150-158. 1077 Mayer, M.P. (2013). Hsp70 chaperone dynamics and molecular mechanism. Trends 1078 Biochem Sci 38, 507-514.

1079 Misselwitz, B., Staeck, O., and Rapoport, T.A. (1998). J proteins catalytically activate Hsp70 1080 molecules to trap a wide range of peptide sequences. Mol Cell 2, 593-603. 1081 Novoa, I., Zeng, H., Harding, H., and Ron, D. (2001). Feedback inhibition of the unfolded 1082 protein response by GADD34-mediated of eIF2a. J Cell Biol 153, 1011- 1083 1022. 1084 Paton, A.W., Beddoe, T., Thorpe, C.M., Whisstock, J.C., Wilce, M.C., Rossjohn, J., Talbot, 1085 U.M., and Paton, J.C. (2006). AB5 subtilase cytotoxin inactivates the endoplasmic reticulum 1086 chaperone BiP. Nature 443, 548-552.

1087 Petrova, K., Oyadomari, S., Hendershot, L.M., and Ron, D. (2008). Regulated association of 1088 misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3. EMBO J 27, 2862- 1089 2872. 1090 Pincus, D., Chevalier, M.W., Aragon, T., van Anken, E., Vidal, S.E., El-Samad, H., and 1091 Walter, P. (2010). BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior 1092 of the unfolded protein response. PLoS Biol 8, e1000415. 1093 Preissler, S., Chambers, J.E., Crespillo-Casado, A., Avezov, E., Miranda, E., Perez, J., 1094 Hendershot, L.M., Harding, H.P., and Ron, D. (2015). Physiological modulation of BiP 1095 activity by trans-protomer engagement of the interdomain linker. Elife 4.

1096 Qi, R., Sarbeng, E.B., Liu, Q., Le, K.Q., Xu, X., Xu, H., Yang, J., Wong, J.L., Vorvis, C., 1097 Hendrickson, W.A., et al. (2013). Allosteric opening of the polypeptide-binding site when an 1098 Hsp70 binds ATP. Nat Struct Mol Biol 20, 900-907.

43 BiP AMPylation

1099 Rahman, M., Ham, H., Liu, X., Sugiura, Y., Orth, K., and Kramer, H. (2012). Visual 1100 neurotransmission in Drosophila requires expression of Fic in glial capitate projections. Nat 1101 Neurosci 15, 871-875.

1102 Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013). Genome 1103 engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308. 1104 Ronda, C., Pedersen, L.E., Hansen, H.G., Kallehauge, T.B., Betenbaugh, M.J., Nielsen, A.T., 1105 and Kildegaard, H.F. (2014). Accelerating genome editing in CHO cells using CRISPR Cas9 1106 and CRISPy, a web-based target finding tool. Biotechnol Bioeng 111, 1604-1616. 1107 Sanyal, A., Chen, A.J., Nakayasu, E.S., Lazar, C.S., Zbornik, E.A., Worby, C.A., Koller, A., 1108 and Mattoo, S. (2015). A novel link between Fic (filamentation induced by cAMP)-mediated 1109 adenylylation/AMPylation and the unfolded protein response. J Biol Chem 290, 8482-8499. 1110 Scorsone, K.A., Panniers, R., Rowlands, A.G., and Henshaw, E.C. (1987). Phosphorylation 1111 of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. J 1112 Biol Chem 262, 14538-14543. 1113 Truttmann, M.C., Wu, Q., Stiegeler, S., Duarte, J.N., Ingram, J., and Ploegh, H.L. (2015). 1114 HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation. J Biol 1115 Chem 290, 9087-9100. 1116 Tsunoda, S., Avezov, E., Zyryanova, A., Konno, T., Mendes-Silva, L., Pinho Melo, E., 1117 Harding, H.P., and Ron, D. (2014). Intact protein folding in the glutathione-depleted 1118 endoplasmic reticulum implicates alternative protein thiol reductants. Elife 3, e03421. 1119 Walter, P., and Ron, D. (2011). The unfolded protein response: from stress pathway to 1120 homeostatic regulation. Science 334, 1081-1086.

1121 Wei, J., Gaut, J.R., and Hendershot, L.M. (1995). In vitro dissociation of BiP-peptide 1122 complexes requires a conformational change in BiP after ATP binding but does not require 1123 ATP hydrolysis. J Biol Chem 270, 26677-26682.

1124 Yang, J., Nune, M., Zong, Y., Zhou, L., and Liu, Q. (2015). Close and allosteric opening of 1125 the polypeptide-binding site in a human Hsp70 chaperone BiP. Structure 23, 1-13. 1126 Yarbrough, M.L., Li, Y., Kinch, L.N., Grishin, N.V., Ball, H.L., and Orth, K. (2009). 1127 AMPylation of Rho by Vibrio VopS disrupts effector binding and downstream 1128 signaling. Science 323, 269-272.

1129 1130

44 BiP AMPylation

1131 Figure Legends:

1132 Figure 1:

1133 Native gel electrophoresis tracks activity state-dependent changes in BiP’s

1134 quaternary structure

1135 A. Schematic representation of BiP’s domain organization in the ATP- and ADP-bound

1136 states. BiP consists of an N-terminal nucleotide binding domain (NBD, pink) and a C-

1137 terminal substrate binding domain (SBD, blue) connected by a hydrophobic interdomain

1138 linker peptide (green). The SBD is composed of a two-layered β-sandwich subdomain

1139 (SBDβ) containing the substrate binding crevice and a helical lid structure (SBDα). In the

1140 ATP-bound conformation the NBD and SBD form extensive contacts, which involves the

1141 linker region, and the SBD is in the open conformation (SBDα extended) allowing for

1142 interactions with substrates (dark blue) at high association and dissociation rates. Upon ATP

1143 hydrolysis to ADP the inter-subunit contacts are lost leading to exposure of the linker,

1144 packing of SBDα against SBDβ, and strong reduction of substrate interaction kinetics.

1145 Cleavage of BiP by the linker-specific protease SubA (scissors) is favored in the ADP-bound

1146 state.

1147 B. Immunoblot of endogenous BiP resolved by native gel electrophoresis. Where indicated

1148 the CHO-K1 cells were exposed to cycloheximide (CHX; 100 µg/mL) or thapsigargin (Tg; 0.5

1149 µM) for the indicated time. The major species visible on the native gel are numbered by

1150 order of descending mobility (I-III) and the monomeric ‘B’ form induced by CHX treatment

1151 and the ‘A’ form detectable in untreated cells are marked. Immunoblots of the same samples

1152 resolved by SDS-PAGE report on total BiP and total eIF2α (which also serves as a loading

1153 control) and phosphorylated eIF2α to reveal the impact of thapsigargin action. Note the

1154 emergence of the ‘B’ form in CHX-treated cells, which is blocked by thapsigargin.

1155 C. BiP immunoblot, as in “A”. Cells were first exposed to cycloheximide to build a pool of the

1156 ‘B’ form of BiP and then challenged with thapsigargin (in the continued presence of

45 BiP AMPylation

1157 cycloheximide). Note the disappearance of the ‘B’ form of BiP and the emergence of BiP

1158 oligomers in the thapsigargin-treated cells.

1159 D. BiP immunoblot of lysates from untreated (-CHX) and cycloheximide-treated (+CHX) cells.

1160 Where indicated the lysates were exposed to the SubA protease that cleaves BiP’s

1161 interdomain linker in vitro, before loading onto the native gel. The cleavage products

1162 detected by the antiserum used on the native gel are noted (CP, upper panel), as are the

1163 full-length BiP (FL) and its substrate binding domains (SBD) on the SDS gel below (the

1164 nucleotide binding domain is not detected by the antiserum). eIF2α serves as a loading

1165 control. Note the resistance of the ‘B’ form of BiP to SubA cleavage.

1166 1167

46 BiP AMPylation

1168 Figure 2:

1169 FICD deletion abolishes BiP modification in cultured cells

1170 A) Schematic illustration of the hamster FICD protein. The transmembrane region (TM), the

1171 two tetratricopeptide repeats (TPR) as well as the Fic-active site are shown. Numbers

1172 represent amino acid positions. The amino acid sequence surrounding the mutations

1173 introduced into the FICD-/- CHO-K1 clone (#49) by CRISPR-Cas9 genome editing are noted.

1174 Both alleles result in premature termination of translation deleting the active site (*).

1175 B) Immunoblots of endogenous BiP from wildtype (wt) CHO-K1 or FICD-/- cell lysates from

1176 which ATP was either depleted by incubation with hexokine and glucose (-ATP, top panel) or

1177 to which ATP (1 mM) had been added (+ATP, bottom panel), resolved by native-PAGE.

1178 Where indicated the cells were exposed to cycloheximide (CHX, 100 µg/ml) or thapsigargin

1179 (Tg, 0.5 µM) for 3 hours before lysis. The major species visible on native gels are numbered

1180 by order of descending mobility (I-III) and the monomeric ‘B’ form induced by CHX treatment

1181 and the ‘A’ form, prominent in ATP-replete lysates of untreated cells, are marked.

1182 Immunoblots of the same samples resolved by SDS-PAGE report on total BiP loaded and on

1183 eIF2α as a loading control. The ATP-supplemented lysates (3 µg/µl protein) were in addition

1184 exposed to SubA (30 ng/µl) for 10 minutes at room temperature prior to separation by SDS-

1185 PAGE and immunodetection of BiP. The intact protein and the substrate binding domain

1186 (SBD), which are detected by the antibodies against a C-terminal epitope of BiP, are

1187 indicated. The asterisk marks a band of unknown identity. Note that neither CHX-dependent

1188 conversion of endogenous BiP into the monomeric ‘B’ form nor the CHX-mediated

1189 resistance of BiP towards proteolytic cleavage by SubA, were observed in FICD-/- cells.

1190 C) Immunoblot of endogenous BiP from wildtype (wt) CHO-K1 and FICD-/- cell lysates

1191 resolved on an isoelectric focusing (IEF) gel. Where indicated the cells have been exposed

1192 to CHX (100 µg/ml) for 3 hours before lysis. Note that the more acidic (‘B’) form of BiP

1193 associated with CHX treatment was absent in FICD-/- cells.

47 BiP AMPylation

1194 D) IEF immunoblot of endogenous BiP from FICD-/- CHO-K1 cells transfected with plasmids

1195 encoding wildtype GST-FICD, the constitutively active GST-FICDE234G or the inactive GST-

1196 FICDE234G-H363A mutant. Mock transfected cells were analyzed as a control. The cells were

1197 treated with CHX (100 µg/ml) for 3 hours before lysis. A pulldown with GSH-Sepharose

1198 beads was performed with the same lysates to analyze expression levels of the plasmid-

1199 encoded GST-FICD fusion proteins. Note that formation of the acidic (‘B’) form of BiP was

1200 restored by expression of catalytically active GST-FICDE234G protein (despite its

1201 comparatively low expression level) but neither by expression of the catalytically inactive

1202 FICDE234G-H363A mutant nor the regulated wildtype enzyme.

1203

48 BiP AMPylation

1204 Figure 3:

1205 AMPylation of purified BiP in vitro recapitulates features of BiP modified in vivo

1206 A) Coomassie-stained native-PAGE gel (left panel) or SDS-PAGE gel (right panel) of

1207 recombinant BiP purified from bacteria (10 µM) exposed to ATP (1.5 mM) in the absence or

1208 presence of recombinant active GST-FICDE234G or inactive GST-FICDE234G-H363A (0.5 µM)

1209 purified from E. coli (45 minutes at 30°C). Where indicated, the interdomain linker-specific

1210 protease SubA (30 ng/µl) was added for 10 minutes. The major species on the native gel are

1211 numbered by order of descending mobility (I-III) and the monomeric ‘A’ and ‘B’ forms of BiP

1212 are indicated. Full-length BiP, the nucleotide binding domain (NBD, also resolved on the

1213 native gel) and the substrate binding domain (SBD) are annotated on the SDS-PAGE gel.

1214 Note the quantitative AMPylation-dependent conversion of BiP into the monomeric ‘B’ form

1215 on native gels and the increased resistance of modified BiP to cleavage by SubA. Also note

1216 that upon incubation with ATP unmodified BiP forms a second slower migrating monomeric

1217 species similar to the ‘B’ form, which likely reflects an alternative (e.g. ATP-bound)

1218 conformation.

1219 B) Immunoblot of an IEF gel of purified hamster BiP19-654 (15 µM) after in vitro AMPylation

1220 with GST-FICDE234G or as a control with inactive GST-FICDE234G-H363A (both at 0.75 µM) in

1221 presence of ATP (1.5 mM) for the indicated times at 30°C followed by treatment with or

1222 without SubA (30 ng/µl) for 10 minutes. The two forms of full-length BiP (‘A’ and the more

1223 acidic ‘B’ form) as well as faint signals that likely represent the unmodified and modified SBD

1224 are indicated.

1225 C) Immunoblot of endogenous BiP from lysates of cycloheximide-treated FICD-/- CHO-K1

1226 cells resolved by IEF-PAGE following exposure in vitro to purified active or inactive FICD (as

1227 in ‘B’ above). Note the conversion of all the detectable BiP to an acidic form in the sample

1228 exposed to active FICD.

49 BiP AMPylation

1229 D) Autoradiograph and Coomassie stain of an SDS-PAGE gel of BiP exposed in vitro to

1230 active GST-FICD-E234G coupled to GSH-Sepharose beads (or GST alone as a control) in

1231 the presence of α-32P-ATP as a substrate. Where indicated the samples were treated further

1232 with increasing concentrations of SubA (0.03 ng/µl, 0.1 ng/µl and 20 ng/µl, lanes 3-5). Note

1233 the confinement of the radiolabel to the SBD fragment of cleaved BiP.

1234

50 BiP AMPylation

1235 Figure 4:

1236 FICD mediated incorporation of a single AMP molecule onto the substrate binding

1237 domain of BiP in vitro

1238 A. Electrospray mass spectra of bacterially expressed hamster BiP (27-654, with a His6-tag)

1239 after reverse phase HPLC purification. The spectra contain protein ions with between 36 and

1240 100 associated protons (the number of protons are indicated for the major species). The

1241 inset shows the data reconstructed onto a true mass scale. The sample in top panel is of

1242 unmodified BiP and that in bottom panel of BiP that had been modified in vitro with GST-

1243 FICD-E234G and ATP.

1244 B. Amino acid sequence of Chinese hamster BiP (with the cleaved signal peptide in lower

1245 case letters). The SubA cleavage site (after L416) is marked by the grey arrow and the

1246 predicted Arg-C and Asp-N AMPylated proteolytic cleavage fragments are delineated by the

1247 colour coded horizontal lines above the protein sequence.

1248 C. LC-MS spectra of peptides derived from recombinant BiP digested with Arg-C or Asp-N

1249 before (“No enzyme”) and after in vitro modification with GST-FICD-E234G and ATP. The m/z

1250 ratio of the signals is displayed in the abscissa and their relative intensity in the ordinate.

1251 The interval of the liquid chromatogram at which the peptides in question eluted is depicted

1252 above each paired sample [tLC (min)]. Note the absence of any signal corresponding to the

1253 doubly-charged non-AMPylated 511-532 Arg-C fragment in the spectrum derived from BiP

1254 after exposure to GST-FICD-E234G and ATP (left-most lower panel) and the absence of

1255 signals corresponding to the doubly-charged AMP modified peptides in the spectra derived

1256 from the Arg-C or Asp-N digests of BiP that had not been exposed to GST-FICD-E234G and

1257 ATP (central and rightmost upper panels).

1258

51 BiP AMPylation

1259 Figure 5:

1260 Mutation of threonine 518 in the substrate binding domain of BiP abolishes its

1261 AMPylation in vitro

1262 A) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of recombinant

1263 bacterially-expressed wildtype BiP and the indicated mutants exposed in vitro to active GST-

1264 FICD-E234G coupled to GSH-Sepharose beads (lanes 2-5) or GST alone as a control (lane

1265 1) in the presence of α-32P-ATP as a substrate.

1266 B) Coomassie-stained native gel of wildtype BiP and the indicated mutants (all at 20 µM),

1267 following exposure to ATP (1.5 mM), GST-FICD-E234G (0.8 µM), both or neither (for 45

1268 minutes at 30°C). Where indicated the samples were further exposed to SubA (30 ng/µl, 10

1269 minutes at room temperature).

1270 C) As in “A” above, with a different set of mutant BiP proteins.

1271 D) As in “B” above, with a different set of mutant BiP proteins. Note that both the T518E and

1272 T518A mutations (in panel “B” above) affect the mobility of the ‘A’ form of BiP and forestall

1273 further changes in mobility by FICD, but only the T518E mutation mimics enzyme-mediated

1274 AMPylation by promoting a ‘B’ form like state partially resistant to SubA cleavage.

1275

52 BiP AMPylation

1276 Figure 6:

1277 Reciprocal loss of unmodified and gain of AMPylated BiP511-532 purified from CHO

1278 cells treated with cycloheximide

1279 A. Schema of the design of the SILAC experiment to quantify relative changes in abundance

1280 unmodified and AMPylated BiP peptides from untreated and cycloheximide (CHX)-treated

1281 wildtype and FICD-/- mutant CHO-K1 cells.

1282 B. LC-MS spectra of unmodified and modified quadruply-charged BiP511-532 peptide from a

1283 SILAC experiment where untreated heavy and cycloheximide-treated light samples from

1284 wildtype CHO-K1 cells were digested by Arg-C. The raw peptide abundance measurements

1285 were normalized to the recovery of a doubly-charged reference peptide, BiP61-74, from the

1286 same SILAC samples (Figure 6-figure supplement 1) to arrive at the normalized ratio of the

511-532 1287 signal in the paired samples (Rnrl). Note that unmodified BiP is depleted and AMPylated

1288 BiP511-532 is reciprocally enriched in cycloheximide-treated wildtype cells.

1289 C. Analysis as in “B” (above) applied to the indicated paired SILAC samples. Note that

1290 unmodified BiP511-532 is depleted by cycloheximide treatment only in wildtype cells and

1291 AMPylated BiP511-532 is only detected in wildtype cells.

1292 These observations were reproduced in a second independent SILAC experiment.

1293

53 BiP AMPylation

1294 Figure 7:

1295 AMPylation of BiP is sensitive to its conformational state

1296 A) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype BiP and the

1297 indicated mutants exposed in vitro to active GST-FICD-E234G coupled to GSH-Sepharose

1298 beads (lanes 2-5) or GST alone as a control (lane 1) in the presence of α-32P-ATP as a

1299 substrate.

1300 B) Bar graph of densitometric quantification of radiolabelled BiP proteins from in vitro

1301 AMPylation reactions as in “A”. The radioactive signals were normalized to the amount of

1302 loaded protein (CBB signal) and the values for wildtype BiP protein were arbitrarily set to 1.

1303 Mean ± SD of three independent experiments are shown.

1304 C) Coomassie-stained native gels of the indicated BiP mutants (all at 20 µM) following

1305 exposure to bacterially expressed GST-FICDE234G (0.8 µM) in presence of 1.5 mM ATP for

1306 the indicated time. Note the reduced ability of BiPE201G to form discrete oligomers in

1307 presence of ATP, which may be due to altered substrate interaction characteristics. Also

1308 note the sharpness of the bands of modified BiPE201G and BiPT229A, which suggest a high

1309 degree conformational uniformity and strongly reduced substrate interactions.

1310 D) Plot of time-dependent accumulation of the ‘B’ form of BiP from experiments as shown in

1311 “C”. Initial values were set to 0% and end-point values were set to 100% for each of the BiP

1312 versions, respectively. Mean ± SD of three independent experiments are shown. Non-linear

1313 regression analysis was performed to determine t1/2max values, which were 9.9 minutes, 4.0

1314 minutes and 2.3 minutes for wildtype BiP, BiPT229A and BiPE201G, respectively.

1315

54 BiP AMPylation

1316 Figure 8:

1317 Functional consequences of BiP AMPylation in vitro

1318 A) Bar diagram of ATP hydrolysis by BiP and BiP AMPylated to completion (BiP-AMP), as

1319 reflected in phosphate release (detected colorimetrically). Samples containing either purified

1320 BiP or BiP-AMP (both at 5 µM) were incubated with 3 mM ATP for 1 hour at 30°C and free

1321 orthophosphate generated by ATP hydrolysis was measured. Samples lacking BiP or ATP

1322 report on assay background. Bar graph shows mean absorbance values ± SD at 612 nm

1323 (A612) of the complex between free orthophosphate and the malachite green dye after

1324 background subtraction of three repeats (n = 3).

1325 B) Bar diagram of Vmax values for basal ATPase activities of unmodified and AMPylated BiP,

1326 derived from experiments as in “A”. Mean values ± SD are shown (n = 3).

1327 C) Measurement of J protein stimulated ATPase activity of unmodified and AMPylated BiP.

1328 Samples of purified BiP or BiP-AMP (both at 1.5 µM) were incubated in presence of 2 mM

1329 ATP with isolated J-domain of ERdj6 (J) at the indicated concentrations for 3 hours at 30°C

1330 and released orthophosphate was detected as in “A”. The control reactions contained 4 µM

1331 of non-functional mutant ERdj6H422Q J-domain (in the critical HPD motif; J*) instead of the

1332 wildtype J-domain. Shown is the J protein-dependent change in ATP hydrolysis rate of BiP

1333 and BiP-AMP relative to their basal ATP hydrolysis rates in absence of J protein (set to 1) of

1334 four experiments (values ± SD, n = 4).

1335 D) Plot of concentration-dependent steady-state binding of substrate peptide by unmodified

1336 or AMPylated BiP. Fluorescence polarization (FP) of 1 µM lucifer yellow-labeled BiP

1337 substrate peptide (HTFPAVLGSC) was measured after incubation with purified BiP or BiP-

1338 AMP at the indicated concentrations for 24 hours at 30°C in presence of 1 mM ADP. Mean

1339 values of a representative experiment performed in triplicates are shown.

1340 E) Plot of time-dependent release of fluorescent-labeled substrate peptide from unmodified

1341 or AMPylated BiP, following injection of 400-fold excess of unlabeled substrate peptide.

55 BiP AMPylation

1342 Fluorescence polarization (FP) signal of lucifer yellow-labeled substrate peptide (1 µM)

1343 bound to BiP or BiP-AMP (both at 40 µM) in presence of 1 mM ADP (as in “D”) was

1344 measured after addition of 400-fold excess of unlabeled substrate peptide (0.4 mM) at t = 0.

1345 The initial values (after background subtraction) were set to 100% and non-linear regression

1346 analysis was performed on the first 60 minutes of peptide competition (inset). Mean values

1347 of a representative experiment performed in triplicates are plotted on the graph. The mean

-1 1348 dissociation rate constants (koff) ± SD for BiP = 0.037 ± 0.006 min and BiP-AMP = 0.212 ±

-1 1349 0.021 min as well as the mean half-lives ± SD (t1/2) for BiP = 19.3 ± 2.9 min and BiP-AMP =

1350 3.3 ± 0.3 min were calculated based on three independent experiments.

1351

56 BiP AMPylation

1352 Figure 9:

1353 Overexpression of active FICDE234G activates the UPR

1354 A) Flow cytometry analysis of CHO-K1 CHOP::GFP UPR reporter cells transiently

1355 transfected with plasmids encoding wildtype FICD, the constitutively active FICDE234G or the

1356 inactive FICDE234G-H363A mutant alongside an mCherry transfection marker. Mock transfected

1357 cells that were treated with the UPR-inducing compound tunicamycin (2.5 µg/ml) for 16

1358 hours before the analysis were included as controls. Note the accumulation of CHOP::GFP-

1359 positive (and mCherry-negative) cells in Q3 in the untransfected tunicamycin-treated

1360 samples and enhanced UPR reporter activation in cells transfected with plasmid encoding

1361 active FICDE234G and a co-expressed mCherry marker (reflected in the large number of

1362 double-positive cells in Q2).

1363 B) Native gel immunoblot of endogenous BiP from lysates (supplemented with 1 mM ATP)

1364 of Flp-In T-REx 293 cells that carry a stable transgene encoding a doxycycline-inducible

1365 form of the active FICDE234G mutant. The cells were treated for the indicated time with

1366 doxycycline (Dox) prior to lysis. The major species visible on the native gel are numbered by

1367 order of descending mobility (I-III) and the monomeric ‘B’ and ‘A’ forms are marked.

1368 Immunoblots of the same samples resolved by SDS-PAGE report on FICDE234G expression,

1369 total BiP loaded and on eIF2α as a loading control. In addition, samples of the lysates were

1370 treated with SubA (30 ng/µl) for 10 minutes at room temperature before separation of

1371 proteins by SDS-PAGE and immunoblotting. Full-length BiP (FL), the nucleotide binding

1372 domain (NBD) and the substrate binding domain (SBD) are indicated. The ratios between

1373 the quantified signals of full-length BiP and total BiP (following SubA cleavage) were

1374 normalized to the value observed in lane 1 (arbitrarily to 1) and are indicated below. The

1375 asterisks mark bands of unknown identity. Note the correlation between FICDE234G

1376 expression, the appearance of the monomeric ‘B’ form of BiP on the native gel as well as the

1377 increasing resistance of BiP towards cleavage by SubA.

57 BiP AMPylation

1378 C) Analysis of Flp-In T-REx 293 cells upon doxycycline-induced expression of inactive

1379 FICDE234G-H363A or active FICDE234G as in “B” above.

1380 D) Proliferation assay with Flp-In T-REx 293 cells upon doxycycline-induced expression of

1381 inactive FICDE234G-H363A or active FICDE234G for the indicated times. Shown are mean values ±

1382 SD relative to uninduced cells (set to 1) of three independent experiments (n = 3).

1383

58 BiP AMPylation

1384 Figure 10:

1385 Over-chaperoning in FICD-deficient cells delays UPR signaling

1386 A) Schematic illustration of the rat FICD protein. Protein domains are highlighted and the

1387 mutations introduced by CRISPR-Cas9 genome editing are presented as in Figure 2A.

1388 B) Isoelectric focusing (IEF) followed by immunoblot of endogenous BiP from wildtype (wt)

1389 and FICD-/- AR42j cell lysates.

1390 C) Native gel immunoblots of endogenous BiP from ATP-depleted wildtype and FICD-/-

1391 AR42j cell lysates

1392 D) Endogenous PERK, IRE1α, BiP and immunoblots of an SDS gel on which lysates of

1393 wildtype and indicated FICD-/- AR42j cells were resolved. The cells were pre-exposed to

1394 cycloheximide (CHX, 100 µg/ml) for 3 hours followed by treatment with 1 mM DTT for the

1395 indicated times. Slower migrating phosphorylated PERK is marked (PERK-P). To detect

1396 phosphorylated IRE1α (IRE1α-P) samples of the same lysates were resolved on a Phos-Tag

1397 gel. Actin served as a loading control. Asterisks mark bands of unknown identity. Note the

1398 delayed phosphorylation (activation) of PERK and IRE1α in the two FICD-/- clones. The

1399 experiment was performed four times with the comparable results.

1400 E) Immunoblots of puromycinylated proteins, PERK, IRE1α, BiP and actin from wildtype and

1401 FICD-/- AR42j cells. Where indicated the cells were pre-treated with CHX (100 µg/ml) for 3

1402 hours followed by washout and exposure to puromycin (Puro, 10 µg/ml) in presence or

1403 absence of the reducing agent DTT (1 mM) for 30 minutes. Puromycin incorporation into

1404 nascent chains (reporting on protein synthesis rates), and PERK-P signals expressed

1405 relative to lane 1 (arbitrarily set to 1) are plotted in the bar graph below (mean values ± SD;

1406 of three independent experiments, n = 3). Asterisk indicates a band of unknown identity.

1407 Note persistent protein synthesis in FICD-/- cells that were exposed to DTT after CHX pre-

1408 treatment (lane 8). Also note the lower basal levels of phosphorylated forms of IRE1α and

1409 PERK in untreated FICD-/- cells.

59 BiP AMPylation

1410 Figure 11

1411 Schema depicting the hypothesized relationship between AMPylation and the BiP

1412 chaperone cycle

1413 FICD-mediated AMPylation on Thr518 allosterically traps BiP in a low substrate-affinity ATP-

1414 like state that is refractory to J protein-mediated stimulation of its ATPase activity. Removal

1415 of the modification by a phosphodiesterase allows BiP to re-join the chaperone cycle

1416 (depicted in the lower portion of the cartoon)

1417

60 BiP AMPylation

1418 Legends to Figure supplements, Supplementary and Source data files:

1419 Figure 2-figure supplement 1:

1420 Time-dependent changes in BiP abundance, BiP ‘B’ form and the fraction of BiP

1421 resistance to cleavage by SubA in cells exposed to the reversible ER stress-inducing

1422 agent 2-deoxy-D-glucose (2-DG)

1423 A. Immunoblots of BiP from lysates of untreated cells or cells exposed to 2-deoxy-D-glucose

1424 (2-DG, 3 mM). Where indicated, the 2-DG was washed out before lysis (in the presence of

1425 ATP). The samples in the top panels were exposed to SubA before denaturing SDS-PAGE.

1426 The migration of the undigested BiP (FL), the nucleotide binding domain (NBD) and

1427 substrate binding domain (SBD) are indicated. The total content of BiP in the sample (before

1428 digestion with SubA) and the eIF2α signal (provided as a loading control) are also shown.

1429 Asterisks mark non-specific bands.

1430 Immunoblots of BiP from the same samples resolved by native-PAGE, before and after

1431 cleavage with SubA are shown below. BiPr denotes SubA-resistant BiP detected on native

1432 gels and the positions of the monomeric A and FICD-dependent ‘B’ form, and BiP oligomers

1433 (II) are indicated.

1434 B. Trace of time-dependent changes in total BiP abundance from the samples in “A”

1435 [normalized to the signal in the untreated (t=0) sample of each genotype], and in the fraction

1436 of BiP digested by SubA from the samples exposed to 2-DG.

() 1437 The fraction digested, was derived from the top panels, in “A”. ()

1438 C. As in “B” but for cells exposed to 2-DG for 8 hours followed by washout.

1439 D. Plot of time dependent changes in the abundance of FICD and RPL27 (60S ribosomal

1440 protein L27, a reference gene) mRNA normalized to PPIA (cyclophilin A) internal control, in

1441 wildtype cells exposed to 2-DG.

61 BiP AMPylation

1442 The observations made in the key time points of this detailed experiment have been

1443 reproduced independently twice.

1444 Note the progressive increase in the fraction of BiP susceptible to cleavage by SubA,

1445 observed in the wildtype cells following exposure to 2-DG and the correlated disappearance

1446 of the ‘B’ form on native-PAGE gels and the correlated re-emergence of ‘B’ form and SubA-

1447 resistant BiP during the 2-DG washout; neither of which are evident in the FICD-/- cells.

1448

1449 Figure 3-figure supplement 1:

1450 Comparison of the differential susceptibility of unmodified and AMPylated BiP to

1451 SubA cleavage in vitro

1452 A) Schema of the experimental design. ATP hydrolysis-deficient BiPT229A protein was

1453 AMPylated in presence of radioactive α-32P-ATP with catalytically active GST-FICDE234G

1454 coupled to glutathione-Sepharose beads. The enzyme-containing beads were removed by

1455 centrifugation and excess of non-radioactive ATP was added to the supernatant to

1456 competitively displace non-covalently bound α-32P-ATP from BiPT229A. Unbound nucleotides

1457 were then removed by passing the sample through a desalting column. Trace amounts (1.5

1458 µg) of the recovered 32P-labeled AMPylated BiPT229A protein were added to excess of

1459 unmodified BiPT229A protein (60 µg; a mass ratio of 40:1) and the combined sample was

1460 supplemented with ATP and treated for 30 minutes with increasing concentrations of the

1461 SubA protease [0.08 to 120 ng/µl] before denaturing SDS-PAGE, Coomassie staining and

1462 autoradiography.

1463 B) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP from samples

1464 described above. In the bottom panel, the Coomassie stain and radioactive signals of the

1465 full-length BiP and the SBD were quantified and normalized to the values in lane 1, which

1466 were set arbitrarily to 1 for full-length BiP and to 0 for the SBD (graph). The Coomassie stain

62 BiP AMPylation

1467 signal reports of the fate of unmodified BiPT229A, whereas the radioactive signals report

1468 exclusively on the modified BiPT229A in the combined sample.

1469 C) A shorter exposure (1 hour versus 8 hours) of the autoradiogram shown in “B”, above. It

1470 reveals the substantial conservation of the radioactive signal emanating from intact BiP

1471 across the time course (the green plot in “B” above), which is obscured by changes in the

1472 band width introduced by the progressive digestion of the unlabeled (and unmodified) intact

1473 BiP in the sample.

1474

1475 Figure 4-figure supplement 1:

1476 Chromatographic profile and reconstructed mass spectrum of modified BiP and BiP

1477 modified in vitro with FICD and ATP

1478 A. Total ion current chromatograms from the reverse-phase separation of unmodified and

1479 AMPylated BiP, respectively. The peak regions denoted by the red arrow were used to

1480 generate the mass spectra shown in “B”.

1481 B. Electrospray mass measurements of intact unmodified and modified BiP, after

1482 reconstruction onto a true mass scale. These data were derived from the ion current

1483 chromatograms presented in Figure 4A. Note the homogeneity of the peak corresponding to

1484 modified BiP, consistent with quantitative modification of BiP by a single AMP molecule. The

1485 ions eluting as a shoulder after the main peaks shown in “A” comprise singly charged non-

1486 proteinaceous species.

1487

1488 Figure 4-figure supplement 2:

1489 Modification of BiP with a single AMP molecule in vivo

1490 A. Electrospray ionization mass spectrum of endogenous BiP eluted from a reverse phase

1491 HPLC column after immunoaffinity purification from wildtype CHO-K1 lysates. The cells were

63 BiP AMPylation

1492 treated with cycloheximide (100 µg/ml) for three hours before lysis to enhance BiP

1493 modification. The spectrum in the upper panel contains peaks for multiply charged protein

1494 ions (with between 36 and 100 associated protons), which were used to reconstruct the

1495 molecular masses of BiP (lower panel). Note that only two major peaks (70,538.3 Da and

1496 70,866.1 Da) were obtained. The difference (327.8 Da) between these reconstructed

1497 masses is consistent with the attachment of a single AMP moiety per BiP molecule in vivo.

1498 B. Experiment as in “A” with BiP isolated from cycloheximide-treated FICD-/- CHO-K1 cells.

1499 Only a single molecular mass was reconstructed (70,0532.2 Da), consistent with the inability

1500 of the mutant cells to AMPylate BiP.

1501 Note that in both experiments the masses of non-AMPylated BiP were slightly larger (59.7

1502 Da and 53.6 Da, respectively) than that predicted of mature endogenous BiP (70,478.6 Da),

1503 which may be explained by other quantitative (likely irreversible) post-translational

1504 modification of BiP (e.g. of a lysine residue).

1505

1506 Figure 4–figure supplement 3:

1507 Evidence for the absence of modification of Ser365 or Thr366 in mono-AMPylated BiP

1508 A. Amino acid sequence of Chinese hamster BiP (with the cleaved signal peptide in lower

1509 case letters) with Ser365 and Thr366 highlighted in red. The SubA cleavage site is marked by

1510 the grey arrow and the predicted proteolytic cleavage fragments generated by the four

1511 endoproteases are delineated by the color coded horizontal lines above the protein

1512 sequence.

1513 B. LC-MS spectra of peptides derived from the indicated digests of recombinant BiP before

1514 (“No enzyme”) and after in vitro modification (that went to completion, see Figure 3B) with

1515 GST-FICD-E234G and ATP. The m/z ratio of the signals is displayed in the abscissa and their

1516 relative intensity in the ordinate. The sequence of the peptide encompassing unmodified

1517 Ser365 or Thr366 is indicated. Note the contrast between the abundance of signal

64 BiP AMPylation

1518 corresponding to the mass of the unmodified peptides encompassing Ser365 or Thr366 in the

1519 samples of AMPylated BiP (lower panel in each of the four pairs) and the absence of the

1520 peptide corresponding in mass to the non-AMPylated 511-532 Arg-C fragment in the

1521 spectrum from the same sample of AMPylated BiP (Figure 4C left-most lower panel).

1522

1523 Figure 6-figure supplement 1

1524 No change detected in abundance of unmodified BiP337-367 purified from CHO cells

1525 treated with cycloheximide

1526 Shown are LC-MS spectra of unmodified quadruply-charged BiP337-367 peptide from a SILAC

1527 experiment in which untreated “heavy” and cycloheximide-treated “light” samples from

1528 wildtype cells or FICD-/- cells were digested by Arg-C. The spectrum of a doubly-charged

1529 reference peptide, BiP61-74, from the same SILAC samples is provided for normalization.

1530 Note the absence of change in abundance of peptides encompassing Thr366 in the SILAC

1531 sample digested with Arg-C.

1532 These observations were reproduced in a second independent SILAC experiment.

1533

1534 Figure 6-figure supplement 2:

1535 Fragmentation spectra of unmodified and AMPylated BiP Arg-C peptide 511-532

1536 pinpoints AMPylation to Thr518

1537 A. High energy collision dissociation (HCD) fragmentation spectra of unmodified and

1538 AMPylated BiP511-532 peptides obtained from Arg-C digests of endogenous BiP (‘1’, upper

1539 panels) immunopurified from cycloheximide-treated wildtype CHO-K1 cells grown in medium

1540 containing “light” arginine (R0 from SILAC sample A in Figure 6A) and unmodified or in vitro

1541 AMPylated recombinant BiP (‘2’, lower panels) purified from bacteria. The most informative

1542 BiP511-532 b and y ions encompassing threonine residues Thr512, Thr525 and Thr527 are

1543 indicated (dotted lines). Note that the signal from unmodified fragment y16, which includes

65 BiP AMPylation

1544 Thr518 (red), is detectable in spectra of unmodified BiP511-532 but is absent in spectra of

1545 AMPylated BiP511-532 (close-up view).

1546 B. The amino acid sequences of the b and y ions identified in BiP511-532 fragmentation

1547 spectra of unmodified and AMPylated endogenous BiP (‘1’) or purified recombinant BiP (‘2’)

1548 as shown in “A” are listed below the sequence of the intact BiP511-532 peptide. Threonine

1549 residues Thr512, Thr525 and Thr527 are indicated in green. Fragment ions highlighted in “A” are

1550 marked in blue. Note the selective absence of fragment ions spanning Thr518 (red) from

1551 spectra of AMPylated BiP511-532.

1552

1553 Figure 7-figure supplement 1:

1554 The isolated BiP substrate-binding domain is not measurably AMPylated by FICD in

1555 vitro

1556 Autoradiograph and Coomassie stain of an SDS-PAGE gel of wildtype BiP and a fusion of

1557 the isolated SBD to Smt3 (Smt3-SBD) following exposure in vitro to active GST-FICD-E243G

1558 coupled to GSH-Sepharose beads (lanes 2-3) or GST alone as a control, (lane 1) in the

1559 presence of α-32P-ATP as a substrate.

1560

1561 Figure 7-figure supplement 2:

1562 Loop 7,8 of the BiP substrate binding domain is destabilized in the ATP-bound

1563 conformation.

1564 The structure of the substrate binding domain (SBD) of human BiP in the apo/ADP state

1565 (PDB 5E86) and ATP state (PDB 5E84) rendered in cartoon form with the loop

518 1566 encompassing Thr (L7,8) in stick diagram. L1,2 and L3,4 delimiting the peptide binding grove,

518 1567 the flanking L5,6 and Thr are indicated for orientation.

518 520 1568 Note that the web of polar interactions stabilizing L7,8 in the ADP state (T side chain to N

1569 amine; T518 side chain to D515 side chain; D515 amine to N520 carboxylate; D515 carboxylate to

66 BiP AMPylation

1570 G519 amine; D515 carboxylate to T518 amine and D515 side chain to G517 carboxylate) is

1571 disrupted in the ATP state; disruption that would be maintained by AMPylation of Thr518.

1572

1573 Figure 9-figure supplement 1:

1574 Overexpression of active FICDE234G induces UPR in FICD-/- cells

1575 Flow cytometry analysis of CHO-K1 FICD-/- CHOP::GFP UPR reporter cells transiently

1576 transfected with plasmids encoding wildtype FICD, the constitutively active FICDE234G or the

1577 inactive FICDE234G-H363A mutant as in Figure 9A.

1578

1579 Figure 10-figure supplement 1:

1580 Undetectable FICD protein in FICD-/- AR42j cell lysates

1581 A) Immunoblot analysis of the sensitivity of anti-FICD antibodies. Indicated amounts of

1582 purified bacterially-expressed mouse FICD104-458 were applied to SDS gels followed by

1583 immunoblotting with chicken anti-FICD antibodies (1:1000) and commercial rabbit anti-FICD

1584 antibodies (1:1000). Note: the detection limit of 10 ng antigen and our inability to detect

1585 FICD by direct immunoblot of lysate of ~5 µL packed cell volume, imply a concentration in

1586 the ER of less than 1 µM.

1587 B) FICD from lysates of wildtype (wt) and FICD-/- AR42j cells was immunopurified with

1588 chicken anti-FICD antibodies (IP 1) or rabbit anti-FICD antibodies (IP 2). The recovered

1589 proteins were resolved on SDS-PAGE gels and FICD was detected by immunoblotting with

1590 rabbit anti-FICD antibodies (IP 1) or with chicken anti-FICD antibodies (IP 2). Note that no

1591 FICD signal was detected in the samples from FICD-/- cell lysates.

1592

67 BiP AMPylation

1593 Figure 10-figure supplement 2:

1594 Absence of FICD does not measurably affect the induction kinetics of the

1595 transcriptional response to unfolded protein stress

1596 A) Schematic illustration of the hamster FICD protein. Protein domains are highlighted and

1597 the mutations introduced by CRISPR-Cas9 genome editing into CHOP::GFP CHO-K1 UPR

1598 reporter cell line are presented (as in Figure 2A).

1599 B-C) Conformation of the mutant phenotype by isoelectric focusing (IEF) and native gel

1600 electrophoresis followed by immunodetection of endogenous BiP (as in Figure 2B-C)

1601 D) Flow cytometry analysis of the CHOP::GFP UPR reporter gene activity in wildtype and

1602 FICD-/- CHOP::GFP CHO-K1 UPR reporter cells upon exposure to tunicamycin (2.5 µg/ml)

1603 for the indicated time.

1604

1605 Supplementary File 1

1606 List of plasmids used.

1607

1608 Figure 8-source data 1

1609 Data from three independent repeats (each performed in triplicates) of the experiment

1610 presented in Figure 8E are shown. The insert on top of each graph shows the absolute

1611 fluorescence polarization (FP) signals in arbitrary units (A.U.) of a reference sample

1612 containing only free fluorescent substrate peptide, which were used to create normalized FP

1613 traces of samples containing BiP + peptide. The initial values (after reference signal

1614 subtraction) were set to 100%. The fit to a single phase decay curve (tabulated here) was

1615 better than to a two phase model. The fit values from the three experiments were used to

1616 calculate the average values for “koff” and the half-lives. Experiment 3 is shown in Figure 8E.

68 BiP AMPylation

1617 Figure 10-source data 1

1618 Data from three independent repeats used for quantification shown in the graph in Figure

1619 10E.

1620

1621 Figure 10-source data 2

1622 Source file of the flow cytometry data used to generate the plot in Figure 10-figure

1623 supplement 2D.

1624

69 Figure 1 A B

SBDĮ (lid)

ATP - - 0.5 h CHX 0.5 h Tg 0.5 h CHX + Tg NBD SBDȕ 2 h CHX 2 h Tg 2 h CHX + Tg III III BiP II II $73 B

(IB: BiP) B I

1DWLYH3$*( I Pi A A

$'3 BiP

ADP eIF2Į

eIF2Į3 6'63$*( merge 1234 5678 SubA

C D

-CHX +CHX 6XE$ ++

III CP

10 min CHX + Tg 30 min CHX + Tg 60 min CHX + Tg 180 min CHX + Tg 180 min CHX II

90 min CHX: + + + + + + CP (IB: BiP) B I 1DWLYH3$*( A III

II 100 kDa B 75 kDa FL

(IB: BiP) I 50 kDa

1DWLYH3$*( A IB: BiP 37 kDa

SBD

6'63$*( 25 kDa BiP 37 kDa eIF2Į 6'6 3$*( eIF2Į 1234 100 51 100 72 123456 )/H,)Į (% of lane 1) Figure 2

AB -ATP Cricetulus griseus FICD wt FICD-/- (#49) (CHO-K1 cells)

H363PFIDGNGRTSR -- TM TPR1&2 E234 CHX Tg CHX Tg core 1 24 42 106 134 139 169 227 285 381 411 458 FIC III

FICD-/- (clone #49) II Allele 1: …EMKR118delKEG(X) GPWLRRCTQ* 12 B Allele 2: …EMKR118indelADQRVLQESDPGRR* (IB: BiP) I

Native-PAGE A C BiP wt FICD-/- (#49) SDS- PAGE eIF2Į - - CHX CHX +ATP pH A wt FICD-/- (#49) B IB: BiP -- CHX Tg CHX Tg

III

II B -/- I D (IB: BiP) FICD (#49) A Native-PAGE A 363 G-H G

234 234 BiP SDS- PAGE eIF2Į GST-FICD-E Mock GST-FICD-E GST-FICD SubA: - + + + + + + pH A 100 kDa 75 kDa BiP B * 50 kDa IB: BiP 37 kDa

GST- (IB: BiP) pulldown FICD SDS-PAGE SBD 25 kDa SDS-PAGE (IB: FICD) 0 123456 Figure 3

A Native-PAGE SDS-PAGE SubA:----++++ ----++++ GST-FICD-E234G-H363A: --+---+- --+---+- GST-FICD-E234G: ---+--+- ---+--+- ATP: -+++ -+++ -+++ -+++

150 kDa III 100 kDa 75 kDa BiP II NBD 50 kDa NBD

CBB B 37 kDa SubA A SBD 25 kDa 20 kDa 12345678 1234567 8 B C FICD-/- (#49) SubA: ------+ + lysate plus GST-FICD-E234G-H363A: +- - - - - +- GST-FICD-E234G: -+-+++++ A 363 1.5 mM ATP (min): 450204545 3 8 45 G-H G pH 234 234

A B GST-FICD-E SBD GST-FICD-E SBD-AMP pH A 12 3 4 5 6 7 8 B IB: BiP IB: BiP D

SubA: -- GST: +---- GST-FICD-E234G: - +++ + 150 kDa 100 kDa 75 kDa BiP

50 kDa

37 kDa

SBD 25 kDa 20 kDa

150 kDa 100 kDa 75 kDa BiP

50 kDa NBD 37 kDa CBB Autoradiograph SubA SBD 25 kDa 20 kDa 1234 5 Figure 4

808.61 827.36 +60 A 4×105 +65 +55 Reconstructed mass = 71,060.1 Da 1185.59 948.64 +50 3×105 1094.36 1293.08 +45 1422.54 2×105 1579.99 +40 BiP

Intensity +36

1×105 1778.07 1984.45

0 750 900 1050 1200 1350 1500 1650 1800 1950

4×105 821.74 803.26 Reconstructed mass = 71,395.6 Da 841.17 +65 +60 +55 3×105 904.84 1099.47 1191.13 992.67 1299.14 +50 2×105 +45 1428.75

Intensity +40 1587.50 +36 BiP-AMP 1×105 1786.12 1984.45

0 750 900 1050 1200 1350 1500 1650 1800 1950 m/z B mkfpmvaaallllcavraEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSN PENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVVEKKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAV VTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHL GGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALSSQHQARIEIESFFEGEDFSETLTRAKFEELNMDL FRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLL DVCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFE IDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGK LSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGPPPTGEEDTSEKDEL Asp-N Arg-C SubA site C

tLC (min) = 14.02 - 14.57tLC (min) = 15.04 - 15.99 tLC (min) = 14.25 - 15.60

BiP511-532 (VTAEDKGTGNKNKITITNDQNR) 1074.55 818.46 100 1209.62 100 100 1316.69 80 80 80

60 60 60 769.77 40 40 40 932.50 1020.52

No enzyme 1238.13 Relative Abundance 20 20 20 875.48 961.14 1119.13 1310.46 0 0 0

1316.69 1074.55 100 916.94 100 100 1374.15 515-528 511-532 BiP + AMP 80 80 BiP + AMP 80 (DKGTGNKNKITITN) (VTAEDKGTGNK

G +ATP NKITITNDQNR) 60 60 60 234

40 40 40

Relative Abundance 1158.50 20 1119.24 20 1402.66 20 818.46 945.45 1310.46 1430.61 973.96

GST-FICD-E 0 0 0 1100 1200 1300 1400 1050 1150 1250 1350 1450 800 850 900 950 1000 m/z m/z m/z

Arg-C Asp-N Figure 5

A B

ATP: - -++ GST-FICD-E234G: -+ -+

wt (ctrl.) wt T518A T525A T527A wt wt wt wt T518A T525A T527A T518A T525A T527A T518A T525A T527A T518A T525A T527A 150 kDa 100 kDa IV BiP 75 kDa III III 50 kDa II II 37 kDa B A A

Autoradiograph 25 kDa

20 kDa

150 kDa CBB 100 kDa SubA 75 kDa BiP

50 kDa

37 kDa CBB NBD 25 kDa B 20 kDa A 12345

CBB CD

ATP: -++ GST-FICD-E234G: - -+ wt (ctrl.) wt T518A T518E T366A R470E R492E V461F 150 kDa 100 kDa wt wt wt 75 kDa BiP T518A T518E T366A T518A T518E T366A T518A T518E T366A

50 kDa IV III III 37 kDa II II

Autoradiograph 25 kDa B A 20 kDa A

150 kDa 100 kDa 75 kDa BiP CBB SubA 50 kDa

37 kDa CBB

25 kDa 20 kDa NBD B 12345678 A

CBB Figure 6

A

FICD+/+ FICD+/+ FICD-/- FICD-/- FICD-/- FICD+/+ R0 R10 R0 R10 R0 R10 “light” “heavy” “light” “heavy” “light” “heavy”

CHX Untreated CHX Untreated CHX CHX

Sample A Sample B Sample C

B Unmodified BiP511-532 AMPylated BiP511-532

Sample A Sample A 607.82 687.58 100 100

90 607.57 90

80 80 605.32 687.83 70 608.07 70 687.33 690.08 60 605.06 60 605.57 50 50 689.83 690.33 40 40 608.32 688.08

Relative Abundance 30 605.82 30 690.58 20 20

10 10

0 0 604.5 605.5 606.5 607.5 608.5 609.5 686.0 687.0 688.0 689.0 690.0 691.0 692.0 m/z m/z CHX Untreated CHX Untreated

Rnrl (UT/CHX) = 1.50 Rnrl (UT/CHX) = 0.67

C Unmodified BiP511-532 AMPylated BiP511-532

Sample C Sample B Sample C 605.32 605.32 690.08 100 100 607.82 100 90 90 607.57 90 689.83 605.06 80 80 605.06 80 690.33 605.57 70 70 605.57 70 608.07 60 607.82 60 60 607.57 50 50 50 608.07 690.58 40 40 40 605.82 605.82 608.32

Relative Abundance 30 30 30 608.32 20 20 20

10 10 10

0 0 0 604.0 605.0 606.0 607.0 608.0 609.0 604.0 605.0 606.0 607.0 608.0 609.0 686.5 687.5 688.5 689.5 690.5 691.5 m/z m/z m/z CHX CHX CHX Untreated CHX CHX FICD-/- FICD+/+ FICD-/- FICD-/- FICD-/- FICD+/+

Rnrl (WT/MUT) = 0.39 Rnrl (UT/CHX) = 0.90 Rnrl (WT/MUT) = ’ Figure 7 A C ATP GST-FICD-E234G (min): -24 6 8 1020 15 45 wt (ctrl.) wt E201G T229A ADDA T37G G226D

150 kDa 100 kDa 75 kDa BiP wt 50 kDa B A 37 kDa

25 kDa

20 kDa

150 kDa 100 kDa 75 kDa BiP E201G B 50 kDa A 37 kDa CBB Autoradiograph

25 kDa

20 kDa

1234567 T229A B B A

6

4

2 ADDA A

AMPylation rel. to wt 0

wt T37G E201G T229A ADDA G226D

D G226D A 120

100

80 CBB

60 wt T229A

‘B’ form (%) ‘B’ form 40 E201G 20

0 0 5 10 15 20 25 30 35 40 45 Time (min) Figure 8

AB

0.4 4 P > 0.0001

0.3 3 M/min) μ 0.2 2 - background (pmol/

612 0.1 1 max A V

0.0 0

ATP BiP BiP

BiP-AMP BiP + ATP BiP-AMP

BiP-AMP + ATP

CD

BiP + J BiP-AMP + J BiP + J* BiP-AMP + J*

1.7 80 ADP 1.6

1.5 60 1.4 1.3 40 (A.U.) 1.2 BiP 20

1.1 background - FP BiP-AMP 1.0 0 Fold change in ATP hydrolysis 01 234 0 10203040 J protein (μM) BiP or BiP-AMP (μM)

E

Peptide

120 ADP 100 100 80 60 80 40

60 20 0 102030405060 40 FP (% of initial) BiP 20 BiP-AMP 0 0 60 120 180 240 300 Time (min) Figure 9

A CHO-K1 CHOP::GFP B FICD-E234G (#9) Mock transfected Dox (h):01033 3 6 24

III

II

* B

(IB: BiP) I A Native-PAGE Tunicamycin

BiP

eIF2Į

50 kDa FICD *

100 kDa SubA / IB: BiP 234 363 75 kDa FL FICD-E G-H A * * SDS-PAGE 50 kDa NBD 37 kDa * SBD 25 kDa 123456 1.00 1.21 1.37 1.33 1.42 1.62 FL/BiP:

FICD C A (#1) A 363 G-H G (#9) G (#31) 234 234 234 FICD-E234G FICD-E parental FICD-E FICD-E Dox (24 h):--++ + - - +

III

mCherry (610 nm) II

B

(IB: BiP) I

CHOP::GFP Native-PAGE A (530 nm)

D BiP 1.2 eIF2Į 1.0 FICD 50 kDa * 0.8 100 kDa SubA (IB: BiP) 75 kDa FL 0.6 *

SDS-PAGE 50 kDa 0.4 NBD 37 kDa 234 363 Cell viability (A.U.) FICD-E G-H A (#1) 0.2 * FICD-E234G (#9) SBD 25 kDa 0 0 5 10 15 20 25 30 35 12345678 1.00 0.84 0.77 0.70 7.18 11.13 7.48 8.34 Doxycycline (h) FL/BiP: Figure 10

ABRattus norvegicus FICD (AR42j cells)

H363PFIDGNGRTSR AR42j TM TPR1&2 E234 wt FICD-/- (#18) core 1 24 42 106 134 139 169 227 285 381 411 458 FIC - - CHX CHX pH FICD-/- (clone #18) A Allele 1: …LEAK107delARG(X) MPSRWTLAS* 8 B 119 Allele 2: …MKRQ delGAK(X)6MPSRWTLAS* IB: BiP FICD-/- (clone #14) Allele 1: …EMKR118delTSSSMPSRWTLAS* 119 Allele 2: …MKRQ delEEG(X)12GPWLRRRPQ*

AR42j C FICD-/- E wt (#14) AR42j - - -/-

CHX CHX wt FICD (#18) Puro Puro Puro Puro Puro Puro Puro Puro +++++ +++

II - - - - DTT DTT DTT DTT

B (IB: BiP) I Native-PAGE A -- -- CHX CHX CHX CHX

150 kDa BiP 100 kDa SDS- PAGE 75 kDa

50 kDa

D IB: Puro AR42j 3 h CHX 37 kDa wt FICD-/- (#18) DTT (min): - 2 5 10 20 30 - 2 5 10 20 30 25 kDa PERK-P 150 kDa PERK 20 kDa *

17 kDa ** IRE1Į-P IRE1Į PERK-P 150 kDa PERK * 75 kDa BiP 50 kDa IRE1Į-P Actin IRE1Į 12345678910 11 12 75 kDa BiP 3 h CHX 50 kDa Actin wt FICD-/- (#14) 1 2345678 DTT (min): - 2 5 10 20 30 - 2 5 10 20 30 PERK-P 8 1.5

PERK-P Puro (rel. to lane 1) PERK 150 kDa Puro * 6 1.0 ** IRE1Į-P 4 IRE1Į 0.5

75 kDa BiP (rel. to lane 1) 2 50 kDa Actin PERK phosphorylation 0 0.0 12345678 12345678910 11 12 Figure 11

Thr518

FICD ?

ATP

ADP

AMP

NEF

BiP J protein Folded protein

Unfolded protein

NEF J