Genetic Editing of SEC61, SEC62, and SEC63 Abrogates Human

bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Genetic editing of SEC61, SEC62, and

2 SEC63 abrogates human cytomegalovirus

3 US2 expression in a signal peptide-

4 dependent manner

7 Anouk B.C. Schuren 1, Ingrid G.J. Boer1, Ellen Bouma1,2, Robert Jan Lebbink1, Emmanuel

8 J.H.J. Wiertz 1,*

9 1Department of Medical Microbiology, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands.

10 2 Current address: Department of Medical Microbiology, University Medical Center Groningen, Postbus 30001, 9700 RB 11 Groningen, The Netherlands

12 *Correspondence: [email protected] (E.J.H.J.W.)

14 Abstract

15 Newly translated proteins enter the ER through the SEC61 complex, via either co- or post-

16 translational translocation. In mammalian cells, few substrates of post-translational SEC62- and

17 SEC63-dependent translocation have been described. Here, we targeted all components of the

18 SEC61/62/63 complex by CRISPR/Cas9, creating knock-outs or mutants of the individual subunits of

19 the complex. We show that functionality of the human cytomegalovirus protein US2, which is an

20 unusual translocation substrate with a low-hydrophobicity signal peptide, is dependent on

21 expression of not only SEC61α, -β, and -γ, but also SEC62 and SEC63, suggesting that US2 may be a

22 substrate for post-translational translocation. This phenotype is specific to the US2 signal peptide.

24 Introduction

25 Up to one-third of all proteins is secreted or expressed in cellular or organelle membranes(Chen,

26 Karnovsky, Sans, Andrews, & Williams, 2010; Wallin & Heijne, 2008). These proteins have been

27 translocated into the endoplasmic reticulum (ER) during or after their translation. Translocation of

28 newly translated proteins into the ER is facilitated by the SEC61 complex, which consists of a

29 multimembrane-spanning SEC61α-subunit associated with smaller SEC61β and -γ subunits(Kalies,

30 Stokes, & Hartmann, 2008). Depending on the mode of translocation, the complex can be

31 complemented by SEC62 and SEC63(Deshaies, Sanders, Feldheim, & Schekman, 1991; A. E. Johnson &

32 van Waes, 1999; Linxweiler, Schick, & Zimmermann, 2017).

34 Translocation of proteins into the ER can occur either co- or post-translationally(Rapoport, 2007;

35 Zimmermann, Eyrisch, Ahmad, & Helms, 2011). The canonical co-translational route is well established

36 in both yeast and mammalian cells. During translation of a signal peptide, the signal recognition

37 particle (SRP) binds the translating ribosome and guides it towards the SRP receptor at the ER

38 membrane. The SRP receptor subsequently interacts with SEC61α, such that the nascent chain is

39 translocated over the ER membrane as translation continues(Song, Raden, Mandon, & Gilmore, 2000).

40 Lateral opening of the SEC61 complex allows for release of translated transmembrane domains(A. E.

41 Johnson & van Waes, 1999).

43 Post-translational translocation remains elusive in higher eukaryotes but is well-studied in yeast.

44 Cytosolic recognition of post-translationally translocated proteins occurs independently of SRP.

45 Instead, cytosolic chaperones of the Hsp70- and Hsp40 families guide fully translated proteins towards

46 the ER(Chirico, Waters, & Blobel, 1988). Signal sequence characteristics determine which mode of

47 translocation is used. Although signal peptides do not share sequence identity, there is overall

48 homology with regard to their polarity and hydrophobicity(von Heijne, 1985). All signal sequences

49 contain an amino-terminal n-region, a central h-region, and a carboxyterminal c-region, containing

50 the cleavage site for removal of the signal peptide. The n- and c-regions contain charged and polar

51 residues, whereas the h-region is hydrophobic(von Heijne, 1985). Post-translational translocation

52 occurs mostly for proteins with modestly hydrophobic signal sequences because they interact less

53 strongly with SRP(Karamyshev et al., 2014; Ng, Brown, & Walter, 1996; Nilsson et al., 2015; Reithinger,

54 Kim, & Kim, 2013; Zheng & Nicchitta, 1999), and for signal sequences with a positive charge in the n-

55 region(Guo et al., 2018). Post-translational translocation has been suggested to act as a back-up

56 mechanism for ER delivery of proteins that fail to translocate via SRP, either because of suboptimal

57 signal sequence functioning(Conti, Devaraneni, Yang, David, & Skach, 2015; Guo et al., 2018; Kriegler,

58 Magoulopoulou, Amate Marchal, & Hessa, 2018), or because of their short length. Proteins shorter

59 than 110 amino acids in length are translocated in a post-translational manner. It is speculated that

60 translation of these short proteins occurs too rapidly to allow for co-translational recognition by SRP,

61 suggesting that they are recognized post-translationally(N. Johnson, Powis, & High, 2013; Lakkaraju et

62 al., 2012).

63 Another feature that influences translocation efficiency is the usage frequency of amino acid codons

64 in the signal peptide. Signal peptides in general contain a high number of sub-optimal codons. A low

65 concentration of tRNAs binding these rare codons slows down translation, allowing more time for the

66 nascent chain to engage SRP(Pechmann, Chartron, & Frydman, 2014; Wang, Mao, Xu, Tao, & Chen,

67 2015; Yu et al., 2016; Zalucki, Beacham, & Jennings, 2009), thereby improving (co-translational)

68 translocation efficiency(Guo et al., 2018). Correspondingly, slowing down translation using a low dose

69 of cycloheximide strongly increases translocation efficiency(Guo et al., 2018), while replacing all sub-

70 optimal codons in a signal sequence by their optimal counterparts, thereby accelerating translation,

71 can lower protein expression at least 20-fold(Zalucki, Jones, Ng, Schulz, & Jennings, 2010).

73 In yeast, post-translational translocation is mediated by an auxiliary complex, containing the

74 Sec61α/β/γ complex extended with Sec62p, Sec63p, Sec71p and Sec72p(Brodsky & Schekman, 1993;

75 Deshaies et al., 1991; Zimmermann et al., 2011). The structure of this complex has recently been

76 solved(Itskanov & Park, 2019; Wu, Cabanos, & Rapoport, 2019). In mammalian cells, the homologous

77 SEC62 and SEC63 have been suggested to play a role in post-translational translocation(Lang et al.,

78 2012). SEC63 is a Hsp40 chaperone which, via its J-domain, interacts with the ER chaperone BiP to

79 provide the driving force for translocation(Lyman & Schekman, 1995). SEC62 provides an alternative

80 for SRP-mediated translocation. The SEC61 complex can switch from SRP-dependent to SEC62-

81 dependent translocation, when SEC62 outcompetes the SRP receptor for SEC63 binding(Jadhav et al.,

82 2015).

84 The human cytomegalovirus (HCMV) protein US2 is implicated in evading immune recognition via

85 degradation of immunoreceptors including HLA class I via ER-associated protein degradation (ERAD)

86 (Hsu et al., 2015; Wiertz, Tortorella, et al., 1996). US2 contains an atypical signal peptide with low

87 hydrophobicity(Gewurz, Ploegh, & Tortorella, 2002), resulting in inefficient translocation that is

88 potentially facilitated by SEC62 and SEC63(Conti et al., 2015). Here, we assessed which mode of

89 translocation is co-opted by US2. We established clonal cell lines with in-frame or out-of-frame indels

90 for each SEC61/62/63 component. We report that SEC61, SEC62 and SEC63 components are essential

91 for translocation of US2, using US2-mediated ERAD of HLA-I as a readout. The weak signal sequence

92 may direct US2 to the SEC62/SEC63 post-translational translocation pathway. This effect is specific to

93 the US2 signal peptide and the signal peptide is sufficient to sensitize a protein to SEC61/62/63 editing.

94 The SEC mutants obtained provide insights into the structural and functional roles of several of these

95 proteins.

97 Results

98 CRISPR/Cas9-mediated editing of all SEC61/62/63 subunits compromises US2

99 translocation

100 The HCMV-encoded immune evasion protein US2 is an unconventional translocation substrate that

101 has a signal sequence with low hydrophobicity. CRISPR/Cas9 technology enabled us to study US2 in

102 the context of genetically mutated SEC61 subunits. US2-mediated HLA class I degradation was used

103 as an indirect readout for US2 translocation. CRISPR single guide RNAs (sgRNAs) targeting subunits of

104 the SEC61 complex were introduced in a clonal U937 cell line expressing HLA-A2-eGFP, US2 and Cas9.

105 As a control, we CRISPR-targeted the ubiquitin E3 ligase TRC8, which is known to be essential for US2-

106 mediated HLA-I downregulation(Stagg et al., 2009).

107 Flow cytometry was used to determine the expression levels of HLA-A2 as a measure of US2 function.

108 As it is known that ER-resident HLA-I that is rescued from degradation can either accumulate in the

109 cytosol or escape to the cell surface(Stagg et al., 2009; van de Weijer et al., 2017), we measured both

110 the total HLA-A2 expression by means of its eGFP-tag, as well as cell surface expression using an HLA-

111 A2-specific antibody (fig. 1). We tested sgRNAs targeting all subunits of the SEC61/62/32 complex and

112 observed HLA-A2 rescue for SEC61α1, SEC61β, SEC61γ, SEC62 and SEC63 (fig. 1), indicating that US2

113 function is disrupted in these cells. These data are in agreement with a genome-wide CRISPR/Cas9

114 library screen we have performed on HLA-I rescue in the presence of US2 (manuscript in preparation).

115 Only SEC61α2 knock-out did not hamper US2 function. Few biochemical studies have been performed

116 on SEC61α2 to date, and it is unclear whether this subunit is in fact part of a functional translocation

117 complex in U937 cells.

118

119 The clear HLA-A2 rescue phenotype at 12 days post-transduction with the SEC sgRNAs (fig. 1) was

120 surprising. Our experience is that knocking out essential ERAD genes, such as p97/VCP and its co-

121 factors Ufd1 and Npl4, yields a minor and transient HLA-I rescue phenotype due to lethality of the

122 knock-out cells and hence loss of the phenotype over time (fig. 2A). By contrast, measuring the SEC61-

123 edited cells over time showed that the HLA-A2 rescue phenotypes were apparent for at least 28 days

124 (fig. 2B), although the effect waned after a peak at 11-14 days post-transduction. The phenotype also

125 seemed specific to the HCMV protein US2, as the activity of another ERAD-inducing HCMV protein,

126 US11(Wiertz, Jones, et al., 1996), was not sensitive to SEC61 editing (fig. 2C), with the exception of

127 SEC63.

128

129 Stable mutant cell lines can be generated for all SEC61 components

130 Since the HLA-I rescue phenotype in SEC61 knock-out cells declined at later timepoints (fig. 2B) we

131 next assessed whether this was caused by cell death due to SEC61 subunit knockout, or by a growth

132 disadvantage of the mutant cells over non-targeted cells in the polyclonal cell population. In the latter

133 case, clonal cell lines with a SEC61 mutation might survive long-term and enable us to study their

134 effect on US2 functioning. For this we sorted single HLA-A2high cells from the polyclonal SEC61/62/63

135 mutant populations by FACS and allowed these to grow out. We were able to establish clonal lines

136 that displayed stable rescue of HLA-I (fig. 3A). CRISPR/Cas9-induced mutations in these clonal cell lines

137 were assessed by sequencing the sgRNA target regions (table S1). The majority of the clones showed

138 in-frame indels in one or both alleles (table S1 and fig. S1), although full genetic knock-outs of SEC61β,

139 SEC61γ and SEC62 were selected with out-of-frame deletions in both alleles. For each SEC61/62/63

140 subunit, two clones were selected for further characterization (marked with ‘1’ and ‘2’ in fig. S2). All

141 clones showed stable rescue of HLA-A2 (fig. 3A), although some displayed a decline in growth rate

142 (data not shown).

143

144 SEC61/62/63 cDNAs revert the HLA-I rescue phenotype of mutant clones

145 To ascertain that the HLA-I rescue phenotypes we observed were specific to the SEC61/62/63 genes

146 that were edited, we expressed sgRNA-resistant cDNAs of the respective genes in the clonal mutant

147 lines and assessed HLA-I levels by flow cytometry. HLA-I rescue phenotypes as well as growth defects

148 were reverted by supplementing with either wildtype or StrepII-tagged cDNAs of the genes that were

149 knocked out (fig. 3B). Expression of the cDNAs was confirmed in Western blot (fig. 3C). In clonal cell

150 lines with a SEC61β, SEC62 or SEC63 mutation, the respective proteins were not detectable by

151 Western blot, suggesting that gene-editing resulted in a full knockout of protein expression. The

152 respective cDNAs could be readily detected when these were introduced. We did not observe a change

153 in SEC61α expression in the mutant clones. To be able to detect the expression of the cDNA that was

154 added, we tagged this cDNA with an additional HA-tag. The lack of phenotype on the SEC61α protein

155 level may be due to the in-frame mutations we identified in the two isolated clones (table S1). Besides

156 the two in-frame SEC61α1-edited genes, we did isolate out-of-frame clones, but these cells were

157 unstable and died within a few weeks (data not shown). This suggests that SEC61α1 is essential for

158 cell survival, which is in agreement with RNAi studies reported previously(Lang et al., 2012). We were

159 unsuccessful to reliably detect SEC61γ by Western blot. Therefore, a StrepII-HA-tagged cDNA was used

160 to detect the SEC61γ cDNA via its HA-tag, similar to the approach used for SEC61α1. Expression of all

161 cDNAs was confirmed in Western blot.

162

163 Genetic editing specifically affects protein expression of the targeted SEC61/62/63

164 component

165 We also tested whether the stability of non-targeted subunits was destabilized by CRISPR/Cas9-

166 mediated editing of single subunits, as we hypothesized that the incomplete and potentially aberrantly

167 assembled SEC61/62/63 may be recognized by protein quality-control mechanisms. As we did not

168 assess the interactions between the SEC61/62/63 subunits, we cannot distinguish between

169 disintegration of the SEC61/62/63 complex and the stabilization of independent subunits. We do

170 however observe that protein expression of non-targeted subunits remains intact, suggesting that the

171 remaining subunits of the complex are not degraded (fig. 4A).

172 In agreement with flow cytometric analysis (fig. 3A), all isolated clones expressed increased levels of

173 HLA-A2-eGFP compared to US2-expressing control cells (fig. 4A, top panel). Endogenous HLA-I rescue

174 was more pronounced than that of the chimeric HLA-A2-eGFP molecule (fig. 4A, bottom part of top

175 panel).

176

177

178 Expression of US2 decreases upon genetic editing of SEC61α1

179 We observed a strong downregulation of full-length US2 in clonal SEC61α1 mutant cells (fig. 4B), which

180 suggests that the HLA-I rescue phenotypes are caused by a defect in US2 expression, rather than

181 impaired ERAD functioning. Preliminary data suggest that a similar reduction in expression of mature

182 US2 can be observed for the other clones (data not shown). The low baseline expression level of US2,

183 even in control cells that were not targeted by CRISPR/Cas9, posed a technical difficulty that did not

184 allow for validating the US2 expression in the other mutant clones. Expression of the transferrin

185 receptor, another transmembrane protein that is translocated by the SEC61 complex, is not affected

186 in our mutant clones, suggesting that this translocation defect is not a general phenomenon.

187

188 The signal peptide determines susceptibility of US2 to SEC61/62/63 editing

189 To be able to detect US2 more effectively, we designed a panel of HA-tagged US2 constructs with

190 engineered signal sequences (figs. 5A, S3 and S4). As a non-glycosylated US2 has been

191 described(Gewurz et al., 2002; Wiertz, Tortorella, et al., 1996), potentially arising from inefficient

192 translocation, and as the signal sequence of a protein influences its translocation efficacy(Kim, Mitra,

193 Salerno, & Hegde, 2002), we tested the US11- and CD8 leader peptides (US11L and CD8L) alongside

194 the original US2L.

195 First, we tested whether the different US2 variants were able to downregulate the HLA-A2-eGFP

196 construct. All US2 variants were equally able to strongly downregulate HLA-A2-eGFP, as was assessed

197 by flow cytometry (fig. 5B). We next transduced these US2-expressing cells with SEC61/62/63-targeted

198 CRISPR sgRNAs (fig. 5C). Overlays of the flow cytometry data from cells with (red) and without (blue)

199 sgRNAs show that only US2L-expressing US2 variants show significant HLA-A2-eGFP rescue upon

200 SEC61/62/63 editing, whereas US2 with a CD8 or US11 leader sequence did not. This phenotype was

201 most pronounced when targeting SEC61α1, SEC62 and SEC63. In control cells lacking US2, a minor

202 reduction of HLA-I expression is observed only upon sgRNA-targeting of SEC61α1 and SEC61γ (fig. 5C,

203 top panels), suggesting that HLA class I expression is also slightly hampered in these cells.

204

205 US11 requires functional SEC61/62/63 upon transfer of the US2 signal peptide

206 We also tested these different signal peptides fused to the HA-tagged HCMV protein US11, which was

207 not sensitive to SEC61/62/63 editing (fig. 2C). To be able to compare the panel of US2 variants to US11,

208 we stably expressed a non-tagged HLA-A2 molecule in U937 cells using lentiviral transduction, as the

209 C-terminal eGFP-tag on the HLA-A2-eGFP construct renders it insensitive to US11-mediated

210 degradation(Cho, Kim, Ahn, & Jun, 2013). We subsequently introduced the various HA-tagged US2 and

211 US11 variants and assessed their potential to downregulate HLA-A2 by flow cytometry (fig. 6A). All

212 US2- and US11 variants were able to downregulate HLA-A2, with US11L- and CD8L-HA-US11 being the

213 most potent (fig. 6A). We observed strong signal peptide-specific differences in the expression levels

214 of the US2- and US11 variants (fig. 6B), which show an inverse correlation with the magnitude of HLA-

215 I downregulation. Subsequently, sgRNAs targeting the SEC61/62/63 subunits were introduced in the

216 six US2- or US11-expressing cell lines, and HLA-A2 expression was assessed by flow cytometry. To be

217 able to compare between cell lines, HLA-A2 rescue was normalized to that in sgRNA-targeted TRC8

218 (for US2) or TMEM129 (for US11) cells, which were used as positive controls. The US2 variants showed

219 an HLA-A2 rescue pattern that was similar to that observed for the HLA-A2-eGFP construct (fig. 5C),

220 with the US2L-variant showing a substantial HLA-A2 rescue effect upon SEC61/62/63 editing, and the

221 US11L- and CD8L-variants being less strongly affected (fig. 6C, top part). Both US11L-HA-US11 and

222 CD8L-HA-US11 retained their ability to downregulate HLA-A2 in the presence of SEC61/62/63 sgRNAs

223 (fig. 6C, bottom part), as was expected from studies performed in fig. 2C. Interestingly, when the US11

224 signal peptide was replaced by that of US2, the US11 protein gained sensitivity to SEC61/62/63 editing

225 (fig. 6C, bottom left). The low expression level of proteins containing the US2L signal peptide (fig. 6B)

226 suggests that this signal peptide indeed functions inefficiently, which may result in increased

227 sensitivity to translocation defects.

228 Taken together, HLA-I degradation by HCMV US2 or US2L-containing US11 is decreased when subunits

229 of the SEC61/62/63 complex are edited by CRISPR/Cas9. Our data suggest that the inferred

230 dependence of US2 on the SEC61/62/63 complex is specific to the inefficient signal peptide it bears.

231

232 Discussion

233 In this study, we assessed the role of SEC61, SEC62, and SEC63 components in HCMV US2 translation.

234 To study the role of the SEC61 complex in US2 functionality, we established clonal cell lines containing

235 mutants of single subunits of the SEC61 complex. Clonal cell lines provide a well-characterized context

236 to study the effect of protein mutations or deletions. Although knockdown of certain SEC61 subunits

237 is possible(Lang et al., 2012) and particular mutations in the complex are compatible with

238 life(Haßdenteufel, Klein, Melnyk, & Zimmermann, 2014; Linxweiler et al., 2017), it was surprising that

239 CRISPR/Cas9-mediated knock-outs or severe mutation of these important genes yielded stable clonal

240 cell lines. These cell lines may be used in a broader context for studying translocation.

241

242 The characterization of the mutant clones allows us to hypothesize how they affect US2 translocation.

243 For the SEC61α1 clones, the mutations are in the cytosolic loop between transmembrane domains 6

244 and 7. Crystallography studies suggest that the residues deleted in our cell lines would normally

245 interact with the ribosome(Voorhees, Fernández, Scheres, & Hegde, 2014), which in our cells might

246 lead to a co-translational translocation defect due to disturbed ribosome interaction. Because SEC61α

247 comprises the actual translocation channel and a full translocation block may be lethal to any cell, we

248 hypothesize that the SEC61α1 mutations must have a mild effect on translocation. By contrast, SEC61β

249 is not essential in yeast(Finke et al., 1996; Görlich & Rapoport, 1993). In line with this, we have been

250 able to create clonal full knockouts of SEC61β. However, SEC61β’s non-essential role in translocation

251 suggests that translocation could still occur in our knock-out cells. We also confirmed the previously

252 reported interaction between SEC61β and US2(Wiertz, Tortorella, et al., 1996) (data not shown). In

253 contrast to SEC61β, SEC61γ is essential in yeast(Falcone et al., 2011). The relatively small HLA-I rescue

254 phenotypes observed upon SEC61γ editing resemble those of essential genes like SEC61α1 and p97,

255 suggesting that SEC61γ is indeed important for cell survival. Alternatively, SEC61γ might simply be less

256 important for US2 function. SEC61γ has however been reported to directly interact with US2(Wiertz,

257 Tortorella, et al., 1996), so mutating it may disturb US2 function and therefore explain the HLA-I rescue

258 we observe. SEC62 has previously been knocked down in mammalian cells(Greiner et al., 2011; Lang

259 et al., 2012), suggesting that it may not be an essential gene for cell survival. We now confirm this with

260 a full knockout clone of SEC62. Interestingly, in SEC62-expressing cells an interaction between US2

261 and SEC62 can be observed (unpublished observation). The 100-150 amino-terminal residues of SEC62

262 are required for its interaction with SEC63, through a positively charged domain in SEC62(Wittke,

263 Dünnwald, & Johnsson, 2000). As our sgRNAs target this N-terminal cytosolic domain, we speculate

264 that even the small in-frame deletion observed in clone 1 (around Cys82) may disturb the interaction

265 with SEC63. The SEC63 sgRNAs we use target the gene within its J-domain. This domain interacts with

266 the ER chaperone BiP(Misselwitz, Staeck, Matlack, & Rapoport, 1999). The highly conserved HPD-motif

267 within this domain however remains intact, suggesting that BiP interaction may still occur. Besides

268 being expressed at lower levels, the edited SEC63 in these cells may be less active because of the

269 mutations in the J-domain. Our findings are in agreement with siRNA-mediated silencing of SEC62 and

270 SEC63(Lang et al., 2012). This contrasts the situation in yeast, where Sec62 and Sec63 are

271 essential(Noël & Cartwright, 2018).

272

273 Our data suggest that a translocation defect of US2 may underlie the rescue of HLA-I in the

274 SEC61/62/63-edited cells, as we observed a downregulation of US2 upon mutating the SEC genes. This

275 translocation defect appears specific to US2, as the transmembrane transferrin receptor and also HLA-

276 I (in US2-lacking cells) are hardly affected. The sensitivity of US2 to mutated SEC61/62/63 might be

277 two-fold: 1) US2 is inefficiently translocated(Gewurz et al., 2002; Wiertz, Tortorella, et al., 1996),

278 making it more sensitive to changes in the translocation process, and 2) as a result of inefficient

279 translocation (even in wildtype cells without SEC61/62/63 editing), US2 is generally expressed at

280 relatively low levels. A further decrease in US2 levels, even when it is minor, may hamper HLA-I

281 degradation when US2 expression falls below a critical threshold. Replacing the signal peptide of US2

282 by that of US11 or CD8 results in increased US2 expression levels. In cells with highly expressed US2

283 (or US11) variants, HLA-I degradation is retained even when SEC61/62/63 is edited. This higher protein

284 expression may lead to an excess of US2 or US11, such that minor changes in protein expression due

285 to translocation defects would potentially not affect HLA-I degradation.

286

287 The US2 leader is less hydrophobic than most signal peptides(Gewurz et al., 2002), while this

288 hydrophobicity is crucial for its association with SRP. The low affinity of US2 for SRP may strongly

289 influence the efficiency of co-translational translocation. The US2 signal peptide does not contain any

290 rare codons, which may result in the translating ribosome having an insufficient time window to

291 engage SRP, thereby lowering the chances of successful translocation even further. Finally, the US2

292 signal peptide also contains a positively charged lysine residue in the n-region, which can divert

293 translation towards the post-translational mode(Guo et al., 2018). The low hydrophobicity, lack of

294 sub-optimal codons and positive charge in the n-region of its signal peptide make US2 a potential

295 substrate for post-translational translocation.

296

297 As post-translational translocation is mediated by SEC62 and SEC63, it was interesting to observe a

298 strong reduction of US2 expression upon CRISPR/Cas9-mediated editing of these genes. In mammalian

299 cells, post-translational translocation is described mainly for protein substrates smaller than 100

300 amino acids, with 120-160-residue proteins translocation co-translationally albeit in an inefficient

301 manner, with SEC62-mediated translocation as a backup mechanism(Lakkaraju et al., 2012). Its 199-

302 amino acid size makes US2 an unlikely candidate for SEC62/SEC63-mediated post-translational

303 translocation. It would however be interesting to test whether its sub-optimal signal peptide directs

304 US2 towards post-translational translocation. A similar phenomenon has been described for

305 preprolactin, a 227-residue model protein for SRP-mediated co-translational translocation. When the

306 preprolactin signal peptide was mutated, such that it was translocated less efficiently, it suddenly

307 interacted with SEC62 and SEC63(Conti et al., 2015).

308

309 Acknowledgements

310 We thank Prof. Tom Rapoport and Prof. Domenico Tortorella for vital discussions about the SEC61

311 mutant cell lines, and Dr. Nicholas McCaul for fruitful discussions about the US2 signal peptide and

312 help with the SignalP data. We would also like to thank the Core Facility for Flow Cytometry in the

313 UMCU for sorting the clonal mutant cell lines. A.B.C.S. was funded by the Graduate Programme of

314 the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organization for

315 Scientific Research; NWO) (project number 022.004.018).

316

317 Figure Legends

318 Figure 1: The SEC61 complex plays a role in US2-mediated degradation of HLA-I

319 CRISPR/Cas9 sgRNAs targeting SEC61 subunits, or control sgRNAs, were added to U937 cells

320 expressing HLA-A2-eGFP and untagged US2. Four sgRNAs were introduced per gene, of which the

321 two most potent are shown. At 12 days post-infection, HLA-A2 expression was assessed by flow

322 cytometry. Total (intracellular and extracellular) HLA-A2 expression was measured by means of its

323 eGFP-tag. Cell surface expression of HLA-A2-eGFP was assessed by an HLA-A2-specific (BB7.2)

324 antibody staining. sgRNA sequences are listed in the materials and methods. All knock-outs of SEC61

325 subunits, with the exception of SEC61α2, rescue HLA-I from US2-mediated degradation.

326

327 Figure 2: CRISPR/Cas9 targeting of SEC61 subunits yields stable abrogation of US2-

328 mediated HLA-I degradation

329 A) Addition of CRISPR/Cas9 sgRNAs targeting essential genes such as p97/VCP or its co-factors Ufd1

330 and Npl4 leads to a minor and temporary rescue of HLA-I expression which is lost over time due to

331 lethality of the knock-out. sgRNAs targeting p97, Ufd1, Npl4, TRC8 (positive control), or an empty

332 vector lacking the sgRNA were added to HLA-A2-eGFP- and US2-expressing U937 cells. Expression of

333 HLA-A2-eGFP was assessed in flow cytometry by means of its eGFP tag at the timepoints indicated.

334 B) Using a similar set-up as in A, sgRNAs targeting SEC61α1, SEC61β, SEC62, SEC63, TRC8 (positive

335 control) or the empty vector control were added to HLA-A2-eGFP- and US2-expressing U937 cells.

336 Targeting these SEC61 subunits yields a stable HLA-I rescue phenotype over at least 28 days. This

337 phenotype is specific to the HCMV protein US2, since US11 (C), another ERAD-inducing HCMV

338 protein is not sensitive to SEC61 knock-out. C) Similar to B, except that U937 cells expressing US11

339 instead of US2 were used.

340

341 Figure 3: SEC61/62/63 mutant clonal lines are stable and interfere with US2-mediated

342 HLA-I expression

343 A) Single cells from fig. 2B were isolated by FACS and two clonal lines were selected per edited gene,

344 based on viability, HLA-I rescue potency (see also figure S1) and sequencing of the sgRNA target sites

345 (table S1). HLA-A2 expression was assessed in these clonal cell lines and compared to parental cell

346 lines without a SEC sgRNA or without US2. Selected clones yield a stable HLA-I rescue phenotype

347 (compare black peaks to non-filled gray peaks). B) sgRNA-resistant wildtype or StrepII-(HA-)tagged

348 cDNAs were transduced in the respective mutant clones and were selected for using Zeocin

349 treatment. Addition of a sgRNA-resistant cDNA reverts the mutant phenotypes, showing that the

350 HLA-I rescue effects are specific. C) Lysates of all selected mutant clones shown in A were

351 immunoblotted and stained against the sgRNA-targeted gene. Transferrin receptor (TfR) was added

352 as a loading control.

353

354 Figure 4: Mutating SEC61/62/63 components lowers expression of mature US2

355 A) Lysates of all selected mutant clones shown in fig. 3 were immunoblotted and stained against HLA

356 class I and all components of the SEC61/62/63 complex. Transferrin receptor (TfR) was added as a

357 loading control. Addition of CRISPR/Cas9 sgRNAs only affects expression of the targeted SEC61

358 subunits, but does not impact the stability of the of other subunits, suggesting that editing single

359 subunits does not result in an unstable SEC61/62/63 complex. B) The cell lines shown in figure 3

360 were lysed in Triton X-100 in an experiment separate from the one shown in A. Lysates were stained

361 with an anti-US2 antibody and an anti-transferrin receptor antibody as a loading control. A decline in

362 US2 expression is observed upon sgRNA-targeting of SEC61α1. Preliminary data shows similar results

363 for the other SEC subunits. Due to low US2 baseline expression, this observation could however not

364 be repeated for these other cell lines.

365

366 Figure 5: The signal peptide determines susceptibility of US2 to SEC61/62/63 editing

367 A) Schematic overview of HA-tagged US2-variants with different signal peptides, as compared to

368 wildtype US2 (top). B) U937 cells expressing HLA-A2-eGFP were transduced with the US2 constructs

369 shown in A. HLA-A2 expression was measured in flow cytometry, by means of either its eGFP-tag and

370 an HLA-A2-specific antibody staining using BB7.2. All HA-tagged US2 variants downregulate HLA-A2

371 efficiently. C)U937 cells expressing HLA-A2-eGFP were transduced with the indicated HA-US2

372 construct or the wildtype (untagged) US2. Subsequently, CRISPR sgRNAs targeting the subunits of

373 the SEC61/62/63 complex were introduced. This polyclonal cell population was subjected to flow

374 cytometry to assess total levels (eGFP, x-axis) and cell-surface levels (antibody stain, Y-axis) of the

375 HLA-A2-eGFP chimera. sgRNAs that were used in this figure are: SEC61α1 sgRNA 2; SEC61α2 sgRNA

376 1; SEC61β sgRNA 2; SEC61γ sgRNA 1; SEC62 sgRNA 2; SEC63 sgRNA 1.

377

378 Figure 6: US11 becomes dependent on functional SEC61/62/63 when fused to the US2

379 signal peptide

380 A) The US2 variants from figure 5A as well as HA-US11 variants containing the three different signal

381 peptides were expressed in U937 cells containing a non-tagged HLA-A2 construct. All HA-tagged

382 US2- and US11-variants downregulate this HLA-A2 molecule, as shown by flow cytometry on cell

383 surface expression of HLA-A2 using the allele-specific BB7.2 antibody. B) Western blot on the cell

384 lines from A. Protein expression levels of HLA-I, US2/US11 (via their HA-tags) or Transferrin receptor

385 (TfR; loading control) were assessed. HLA-I expression levels reflect the flow cytometry data shown

386 in A. The expression levels of US2 and US11 are affected by their signal peptides, with US2L leading

387 to a low, US11L to an intermediate and CD8L to a high expression level. C) Relative wildtype HLA-A2

388 expression in the presence of SEC61/62/63 sgRNAs. This experiment was set up similarly to figure

389 5B, albeit with the untagged HLA-A2 construct. HLA-A2 expression was assessed at the cell surface

390 using an HLA-A2-specific antibody. Flow cytometry data is summarized in bar graphs showing the

391 HLA-A2 expression level (based on mean fluorescence intensity). HLA-A2 fluorescence intensity is

392 shown relative to the positive control (TRC8 for US2, or TMEM129 for US11) to allow comparison

393 between graphs.

394

395 Supplementary table 1: Sequence analysis of clonal mutant SEC61/62/63 cell lines

396 From all SEC61/62/63 mutant clones, genomic DNA was isolated and the sgRNA-target site was

397 amplified by PCR. Editing of sgRNA target sites was analyzed by standard Sanger DNA sequencing.

398 Only the two selected clones per target gene are shown. SEC61β was edited at the 3’ end of the

399 gene, resulting in a deletion of the C-terminus. A large part of this deletion was in the DNA encoding

400 the 3’ UTR, and did therefore not impact the protein directly. The large insertions observed in SEC62

401 clone 1 and SEC63 clone 2 are shown only partially. The remaining part of these insertions are listed

402 below the table.

403

404 Supplementary figure 1: Overview of mutated amino acid residues in clonal SEC-

405 targeted cell lines

406 Topological view of human SEC61α, -β, and -γ, and SEC62 and -63. Mutated residues are color-coded

407 according to the description in the figure. Topology images are derived from Protter

408 (wlab.ethz.ch/protter).

409

410 Supplementary figure 2: Protein expression of the targeted SEC61/62/63 subunits in

411 clonal mutant cell lines

412 Immunoblotting was performed for all clonal mutant cell lines generated. Two clones were selected

413 for each target gene, based on non-detectable protein levels in Western blot, the level of HLA-I

414 rescue, or sequencing of the sgRNA target sites in the genomic DNA (table S1). Selected clones are

415 indicated by ‘1’ and ‘2’. Panel A) shows clonal SEC61α1-targeted cells; panel B) those for SEC61β; C)

416 SEC61γ; D) SEC62; E) SEC63. SEC61γ clones were selected based on their HLA-I rescue phenotype.

417

418 Supplementary figure 3: Amino acid and codon properties of the US2-, US11- and CD8

419 signal peptide

420 Signal peptide properties of the US2-, US11-, and CD8 leader respectively. On the X-axis, the amino

421 acids and corresponding codons of the signal peptides are shown. The residues are color-coded in

422 the bar graph to show their characteristics with regards to hydrophobicity, polarity or charge. As

423 multiple codons exist for most amino acids, the height of the bars indicates the frequency at which

424 these codons are used (per 1000 codons). These frequencies were derived from

425 https://www.biologicscorp.com/tools/CodonUsage. A cut-off was set at 1% (10 out of 1000 codons),

426 below which the codons are indicated as rare(Kane, 1995).

427

428 Supplementary figure 4: The US2 signal peptide has weak signal peptide

429 characteristics

430 The N-terminal portions of wildtype US2 or the HA-tagged US2 versions were analyzed using the

431 hidden Markov model (HMM) in SignalP prediction software. This model calculated the probability

432 of the submitted sequence being a signal peptide, including the n-, h-, and c-regions and the

433 predicted cleavage site. For wildtype US2, the signal peptide and part of the protein are shown. For

434 the HA-tagged variants, the signal peptide, HA-tag and the same n-terminal part of the US2 protein

435 are shown. The US2 signal peptide has a low score in this prediction model, suggesting that it bears

436 few of the characteristics of a signal peptide. The HA-tag downstream of the US2 leader may affect

437 signal sequence cleavage, but it is unclear how this impacts the overall HMM score of the signal

438 peptide.

439

440 Materials & methods

441

442 Cell culture

443 Human monocytic U937 cells (ATCC) and human embryonic kidney 293T cells (ATCC) were cultured in

444 RPMI 1640 medium (Gibco), supplemented with 5-10% fetal calf serum (BioWest), 100 U/ml

445 Penicillin/Streptomycin (Gibco) and 2 mM Ultraglutamine-1 (Gibco).

446

447 Lentivirus production and infection

448 The day before virus production, 65,000 293T cells were seeded per well in 24-wells plates. For virus

449 production, 250 ng lentiviral vector was co-transfected with a mix of third-generation lentivirus

450 packaging vectors (250 ng total) using Mirus LT-1 transfection reagent (Mirus Bio LLC). Three days

451 post-transfection, the supernatant was harvested and stored at -80 °C.

452 For lentiviral transduction, 100 μl virus-containing supernatant supplemented with 8 μg/ml polybrene

453 (Santa Cruz Biotechnology) was added to 30,000 cells. The cells were spin-infected at 1,000G for 1.5

454 hours at 33 °C.

455

456 Generation of clonal knockout cell lines

457 To generate knock-out cell lines, U937 cells with stable expression of HLA-A2-GFP, HCMV US2 and S.

458 pyogenes Cas9 (Addgene #52962) were transduced with a lentiviral vector containing a CRISPR/Cas9

459 guideRNA (gRNA) and a puromycin resistance gene. Target sequences of the respective CRISPR sgRNAs

460 are shown below (table 1). Three days post transduction, cells were subjected to 2 μg/ml puromycin

461 to select successfully transduced cells. Single HLA-A2-GFP+ cells were FACS-sorted by an Aria3 cell

462 sorter into 96-wells plates containing RPMI 1640 medium supplemented with 50% FCS and allowed to

463 recover. The knock-out status was confirmed by flow cytometric analysis of HLA-I expression,

464 sequencing of the sgRNA target region (table S1), and addback experiments of the respective cDNAs.

465

Target gene Target sequence + PAM

SEC61α1 (gRNA 1) TGTTGTACTGGCCACGGTAGCGG

SEC61α1 (gRNA 2) ATCAAGTCGGCCCGCTACCGTGG

SEC61α1 (gRNA 3) ATCTCTCCTATTGTCACGTCTGG

SEC61α1 (gRNA 4) CTGAAGACATGATCCCAAACAGG

SEC61α2 (gRNA 1) TATGTCATGACGGGGATGTATGG

SEC61α2 (gRNA 2) CCCCGTCATGACATACACAATGG

SEC61β (gRNA 1) TAGTGGCCCTGTTCCAGTATTGG

SEC61β (gRNA 2) GTAGAATCGCCACATCCCCCCGG

SEC61β (gRNA 3) GACAGTGGATCCCGCCGCCCGGG

SEC61β (gRNA 4) GCACTAACGTGGGATCCTCATGG

SEC61γ (gRNA 1) TGTAAAGGACTCCATTCGGCTGG

SEC61γ (gRNA 2) GCATCTTTTAACCAGCCGAATGG

SEC62 (gRNA 1) CTGTGGTTGACTACTGCAACAGG

SEC62 (gRNA 2) GTAGTCAACCACAGACTCCCTGG

SEC62 (gRNA 3) AACCCGGTGACCCATCATATTGG

SEC62 (gRNA 4) CACCAATATGATGGGTCACCGGG

SEC63 (gRNA 1) GTGATGAGGTTATGTTCATGAGG

SEC63 (gRNA 2) TTGGTATTCTCGGTCTGTTTTGG

SEC63 (gRNA 3) TCCCGGCGACATACTACCTCTGG

SEC63 (gRNA 4) TGTTCCCACTGTCATCGTACTGG

TRC8 AGGAAGATGACAGGCGTCTTGG

TMEM129 GCACACGGCGAACACCAGATAGG

P97 GAGGCGCGCGCCATGGCTTCTGG

Npl4 GATCCGCTTCACTCCATCCGGGG

Ufd1 GATGGAGACCAAACCCGACAGAGG

466

467 Antibodies

468 Primary antibodies used for flow cytometry were: PE-conjugated mouse anti-HLA-A2 (clone BB7.2, BD

469 Pharmingen, no. 558570) and human anti-HLA-A3 mAb (clone OK2F3 LUMC, Leiden, The Netherlands).

470 As secondary antibody for flow cytometry we used PE-conjugated goat anti-human IgG + IgM (H+L)

471 (F(ab')2) (Jackson Immunoresearch, no. 109-116-127).

472 For immunoblotting we used the following primary antibodies: mouse anti-HLA-I HC HCA2 mAb,

473 mouse anti-Transferrin receptor mAb (clone H68.4, Invitrogen, no. 13-6800,), rat anti-HA-tag mAb

474 (clone 3F10, Roche, no. 11867423001), rabbit anti-SEC61A mAb (Abcam, ab183046 [EPR14379]),

475 rabbit anti-SEC61B pAb (Abcam, ab15576), rabbit anti-SEC61G pAb (Abcam, ab16843), rabbit anti-

476 SEC62 pAb (Abcam, ab16843), mouse anti-SEC63 pAb (Abcam, ab68550).

477 Secondary antibodies for immunoblotting were: HRP-conjugated goat anti-mouse IgG (Jackson

478 Immunoresearch, no. 211-032-171), HRP-conjugated goat anti-rat IgG (Jackson Immunoresearch, no.

479 112-035-175), HRP-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, no. 115-035-174).

480

481 Plasmids

482 The C-terminally GFP-tagged HLA-A2 construct was expressed from a lentiviral pHRSincPPT-SGW

483 vector (kindly provided by Dr. Paul Lehner and Dr. Louise Boyle, University of Cambridge). Wildtype

484 HCMV US2 was expressed from an EF1A promoter in a lentiviral plasmid. As this plasmid did not

485 contain a resistance gene for antibiotics selection, US2+ cells were FACS-sorted as described before

486 (Chapter 4). A lentiviral vector containing an EFS promoter-driven codon-optimized S. pyogenes Cas9

487 fused to a nuclear localization sequence was purchased via AddGene (lenti-Cas9-Blast, plasmid

488 #52962). This plasmid also contains a Blasticidin resistance cassette. CRISPR sgRNAs were cloned

489 downstream of a human U6 promoter in a BsmBI-linearized pSicoR lentiviral vector also containing an

490 EFS (EF1A short) promoter driving expression of a puromycinR-T2A-Cas9Flag cassette. cDNAs were

491 expressed from an EF1A promoter on a bidirectional lentiviral vector. This vector also contains a

492 human PGK promoter driving expression of a Zeocin resistance gene.

493

494 Flow cytometry

495 Cells were washed in PBS (Gibco) containing 0.5% BSA and 0.02% sodium azide and subsequently fixed

496 15 min in 0.5% formaldehyde. Cells were stained with the antibodies listed above. Flow cytometry was

497 performed on a FACSCanto II (BD Bioscience) and data was analyzed using FlowJo software.

498

499 Immunoblotting

500 Cells were lysed in Triton X-100 lysis buffer (1% Triton X-100 in TBS, pH 7.5) containing 10 μM

501 Leupeptin (Roche) and 1 mM Pefabloc SC (Roche). Lysates were spun down at 18,000g for 20 min at 4

502 °C to remove cellular debris. After addition of Lämmli sample buffer containing DTT, lysates were

503 boiled 5 min at 95 °C and stored at -80 °C until use. SDS-PAGE was performed on 12% polyacryl-amide

504 gels, after which the proteins were transferred to a PVDF membrane (Immobilon-P, Millipore). Primary

505 antibodies were incubated overnight at 4 °C in 1% milk. The secondary antibodies were incubated for

506 1 hour at room temperature. Each antibody was washed off three times in PBS-T (PBS containing

507 0.05% Tween20 (Millipore)). Protein bands were detected by incubation with ECL (Thermo Scientific

508 Pierce) and exposed to Amersham Hyperfilm ECL films (GE Healthcare).

509

510 Co-immunoprecipitation

511 Cells were lysed in Digitonin lysis buffer, containing 1% Digitonin (Calbiochem) in 50 mM Tris-HCl, 5

512 mM MgCl2 and 150 mM NaCl, at a pH of 7.5. This buffer was supplemented with 10 μM Leupeptin

513 (Roche) and 1 mM Pefabloc SC (Roche). Lysates were incubated on ice for 30 min, after which they

514 were spun down at 18,000g for 20 min at 4 °C to remove cellular debris. The supernatant was

515 incubated overnight with StrepTactin beads (GE Healthcare) on a rotary wheel at 4 °C. The samples

516 were washed 4 times with 0.1% Digitonin lysis buffer and eluted with d-Desthiobiotin (2.5 mM d-

517 Desthiobiotin, 100 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA, at a pH of 8.0). 0.45 μM SpinX

518 columns (Corning Costar) were used to collect the eluate. After addition of Lämmli sample buffer

519 with DTT, samples were boiled 5 min at 95 °C and stored at -80 °C until further use. Immunoblotting

520 was performed as described above.

521

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652 Zheng, T., & Nicchitta, C. V. (1999). Structural determinants for signal sequence function in the mammalian endoplasmic

653 reticulum. J Biol Chem, 274(51), 36623–36630. Retrieved from http://www.jbc.org/content/274/51/36623.full.pdf

654 Zimmermann, R., Eyrisch, S., Ahmad, M., & Helms, V. (2011). Protein translocation across the ER membrane. Biochimica et

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656

657

sgSEC61α1 sgSEC61α2 sgSEC61β sgRNA: 2 3 1 2 3 4

49,5% 3,38% 1,95% 1,45% 25,3% 11,5%

sgSEC61γ sgSEC62 sgSEC63 1 2 3 4 1 3

13,2% 4,86% 52,1% 56,9% 30,7% 61,8%

sgTRC8 sgEV

91,4% 1,00% HLA-A2-eGFP (cell surface) HLA-A2-eGFP

HLA-A2-eGFP total (eGFP) bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 2: CRISPR/Cas9 targeting of SEC61 subunits yields stable abrogation of US2-mediated HLA-I degradation A

100 7 dpi 10 dpi 80 14 dpi cells + 60 18 dpi

40 21 dpi

20 % HLA-A2-GFP

0 sgRNA: p97 Ufd1 Npl4 EV ctrl TRC8

B 100 US2

80 7 dpi 11 dpi cells + 60 14 dpi 18 dpi 40 28 dpi 20

% HLA-A2-GFP 8 0 sgRNA: 121212121212

TRC8 EV ctrl SEC62 SEC63 SEC61β SEC61α1

C 100 US11

80 7 dpi 11 dpi cells + 60 14 dpi 40 18 dpi 28 dpi 20

% HLA-A2-GFP 8 0 sgRNA: 121212121212

EV ctrl SEC62 SEC63 SEC61β TMEM129 SEC61α1 bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 3: SEC61/62/63 mutant clonal lines are stable and interfere with US2-mediated HLA-I expression sgSEC61α1 sgSEC61β sgSEC61γ A clone 1 clone 2 clone 1 clone 2 clone 1 clone 2

sgSEC62 sgSEC63 clone 1 clone 2 clone 1 clone 2

– US2 + US2 – sgRNA + US2 + sgRNA HLA-A2-eGFP cell surface

B sgSEC61α1 sgSEC61β sgSEC61γ clone 1 clone 2 clone 1 clone 2 clone 1 clone 2

wt cDNA

St2 cDNA

sgSEC62 sgSEC63 clone 1 clone 2 clone 1 clone 2

wt cDNA + US2 – sgRNA SEC sgRNA + EV ctrl SEC sgRNA + cDNA St2 cDNA

HLA-A2-eGFP cell surface C sgSEC61α1 sgSEC61α1 cl.1 cl.2 sgSEC61β cl.1 sgSEC61β cl.2 sgSEC61γ cl.1 sgSEC61γ cl.2

cDNA: WT St2HAEV WT St2HAEV cDNA: WT St2 EV WT St2 EV cDNA: WT St2HAEV WT St2HAEV 100 150 Tfr 100 Tfr 100 75 75 Tfr SEC61α 15 SEC61 50 20 SEC61β γ (α-HA) 15 10 (α-HA) 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

sgSEC62 cl.1 sgSEC62 cl.2 sgSEC63 cl.1 sgSEC63 cl.2 cDNA: cDNA: WT St2 EV WT St2 EV WT St2 EV WT St2 EV 150 100 Tfr 100 Tfr 75 75 SEC62 150 50 100 SEC63 1 2 3 4 5 6 1 2 3 4 5 6 bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 4: Mutating SEC61α1 lowers expression of US2

1 A α β γ B α1 sgSEC61sgSEC61sgSEC61sgSEC62sgSEC63 sgSEC61 US2: -+++++++++++ US2: - + + + clone: --1212121212 clone: - - 1 2 100 HLA-A2-eGFP 25 75 20 US2 50 endog. HLA class I 37 150 Tfr 50 SEC61α 75 37 1 2 3 4 15 SEC61β 75 50 SEC62

100 75 SEC63 150 100 Tfr 75 1 2 3 4 5 6 7 8 9 10 11 12 bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 5: The signal peptide determines susceptibility to SEC61/62/63 editing

A B US2L- US11L- CD8L- HA-US2 HA-US2 HA-US2

US2L US2 total (eGFP) US2L HA US2

US11L HA US2

cell surface CD8L HA US2

HLA-A2-eGFP expression

no US2 US2 wildtype HA-US2 variants

C sgRNA: SEC61α1 SEC61α2 SEC61β SEC61γ SEC62 SEC63

no US2

US2L-US2 (WT)

US2L-HA-US2

US11L-HA-US2

CD8L-HA-US2 HLA-A2-eGFP (cell surface) HLA-A2-eGFP HLA-A2-eGFP (total) – sgRNA + SEC sgRNA bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 6: US11 becomes dependent on SEC61/62/63 when fused to the US2 signal peptide

A B HA-US2 HA-US11 US2L US11L CD8L

- US2L US11L CD8L US2L US11L CD8L HA-US2 100 Tfr 37 US11 α-HA 25 US2 HA-US11 20 50 HLA class I HLA-A2 wildtype 37 1 2 3 4 5 6 7 – US2/US11 + US2/US11 C US2L-HA-US2 US11L-HA-US2 CD8L-HA-US2

1 1 1

0.5 0.5 0.5

0 0 0

TRC8 TRC8 TRC8 EV ctrl SEC62SEC63 EV ctrl SEC62SEC63 EV ctrl SEC62SEC63 SEC61SEC61β γ SEC61SEC61β γ SEC61SEC61β γ SEC61SEC61α1 α2 SEC61SEC61α1 α2 SEC61SEC61α1 α2

US2L-HA-US11 US11L-HA-US11 CD8L-HA-US11

1 1 1

0.5 0.5 0.5

relative HLA-A2 wildtype expression 0 0 0

EV ctrl SEC62SEC63 EV ctrl SEC62SEC63 EV ctrl SEC62SEC63 SEC61SEC61β γ SEC61SEC61β γ SEC61SEC61β γ TMEM129SEC61SEC61α1 α2 TMEM129SEC61SEC61α1 α2 TMEM129SEC61SEC61α1 α2

sgRNA bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary table 1: Sequence analysis of clonal mutant SEC61/62/63 cell lines

gene gRNA allele target sequence incl. PAM indels SEC61A1 WT CTGCCAATCAAGTCGGCCCGCTACCGTGGCCAGTA...TGTCCAACCTTTATGTCATCT 1 CTGCCAATCAAGTCGGCCCG------TGGCCAGTA...TGTCCAACCTTTATGTCATCT -6 cl.1 2 2 CTGCCAATCAAGTCGGCCCG------...------TTTATGTCATCT -90

SEC61A1 WT CTGCCAATCAAGTCGGCCCGCTACCGTGGCCAGTACAACACCTATCCCATCAAGCTCTT 1 CTGCCAATCAAGTCGGCCCG------TGGCCAGTACAACACCTATCCCATCAAGCTCTT -6 cl.2 2 2 CTGCCAATCAAGTCGGCCCGC------TACAACACCTATCCCATCAAGCTCTT -12

SEC61B WT TCTAGTGGCCCTGTTCCAGTATTGGTTATGAGTC...TGAGGAATCAGTTTTTTTCTAT 1 TCTAGTGGCCC------TGGTTATGAGTC...TGAGGAATCAGTTTTTTTCTAT -11 cl.1 1 2 TCTAGTGGCCCTGTT------...------TTTTTCTAT -232*

SEC61B WT TATTTCTAGTGGCCCTGTTCCAG------TATTGGTTATGA...CAGTTTTTTTC 1 TATTTCTAGTGGCCCTGTTCCAGGGGTTATGGTTATTGGTTATGA...CAGTTTTTTTC +10 # cl.2 1 2 TATTTCTAGTGGCCCTGTTCCAG------...-----TTTTTC -226

SEC61G WT ATCTTTTAACCAGCCGAATGGAGTCCTTTACAAACTGCCGACTTGGCTCAACAAACTGC 1 ATCTTTTAACCAGCCGA------CTTGGCTCAACAAACTGC -24 cl.1 1 2 ATCTTTTAACCAGCC-A------AAACTGCCGACTTGGCTCAACAAACTGC -15

SEC61G WT TTCTATCAGGTTTAGTGCATCTTTTAACCAGCC-GAATGGAGTCCTTTACAAACTGCCG 1 TTCTATCAGGTTTAGTGCATCTTTTAACCAGC--GAATGGAGTCCTTTACAAACTGCCG -1 cl.2 2 2 TTCTATCAGGTTTAGTGCATCTTTTAACCAGCCCGAATGGAGTCCTTTACAAACTGCCG +1 3 TTCTATCAGGTTTAGTGCATCTTTTAA--A-----AAAGGAGTCCTTTACAAACTGCCG -6

SEC62 WT AACCAGGGAGTCTGTGGTTGACTACTGC------AA---...---CAGGTACTGTTT 1 AACCAGGGAGTCTGTGGTTGACTACTGGTTGAAATAAA---...---CAGGTACTGTTT +8 $ cl.1 1 2 AACCAGGGAGTCTGTGGTTGACTACTGC------AATAA...AAACAGGTACTGTTT +27

SEC62 WT AAGCTTTATTTACAACCAGGGAGTCTGTGGTTGACTACTGCAACAGGTACTGTTTATTT 1 AAGCTTTATTTACAACC----AGTCTGTGGTTGACTACTGCAACAGGTACTGTTTATTT -4 cl.2 2 2 AAGCTTTATTTACAACCAGG--GTCTGTGGTTGACTACTGCAACAGGTACTGTTTATTT -2

SEC63 WT GCATAAGCTTTTGCTATCCTCATGAACATAACCTCATCACCTCTTTATCTGGATGATAT 1 GCATAAGCTTTTG--AT----AT------CCTCATCACCTCTTTATCTGGATGATAT -14 cl.1 1 2 GCATAAGCTTTTGCTATCCTCAT------AACCTCATCACCTCTTTATCTGGATGATAT -6

SEC63 WT GCTATCCTCATG------...------AACATAACCTCATCACCTCCTTT 1 GCTATCCTCATG------...------CACCTCCTTT -13 & cl.2 1 2 GCTATCCTCATAACCTAACCTC...CCTATCCTCATAACATAACCTCATCACCTCCTTT +57

* -78 in coding sequence; rest in 3’ UTR # -74 in coding sequence; rest in 3’ UTR $ rest of insertion = TAATAAAATAATAATAAAAAT & rest of insertion = ATAAGGATAGCTTTTGCTATCCTCATAACCTTATAA bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary figure 1: Overview of mutated amino acid residues in clonal SEC- targeted cell lines 760 750 740 730 710 720 700 690 680 PTMs variants disulfide bonds signal peptide N-term: UniProt TMRs: UniProt 660 670 650 640 620 630 610 600 68 590 580 60 570 Clone 1 Allele 1 = in-frame deletion of R12-I19 (blue + red) Allele 2 = in-frame deletion of V15-I19 (red) + R20W point mutation Clone 2 Allele 1 = out-of-frame deletion starting at R20 (yellow) Allele 2 = out-of-frame insertion starting at R20 (yellow) 560 550 40 50 30 20 10 SEC61G 530 540 520 510 490 500 480 470 460 440 450 430 420 400 410 390 380 370 360 350 340 330 96 Clone 1 Allele 1 = V71-V73 deleted, thereafter out-of-frame Allele 2 = full deletion of C-term, starting at P72 Clone 2 Allele 1 = out-of-frame insertion after P72 Allele 2 = full deletion of C-term, starting at V73 90 80 310 320 60 70 Clone 1 Allele 1 = out-of-frame deletion starting at V140 (red) Allele 2 = in-frame deletion of M141-F142 (yellow) Clone 2 Allele 1 = out-of-frame deletion starting at D138 (blue) Allele 2 = in-frame insertion of 19 a.a. after F142 (arrowhead) Gray = J-domain 300 290 40 30 20 50 10 280 270 260 250 SEC61B 200 170 180 160 150 190 240 140 210 220 230 120 130 110 100 90 80 70 60 10 20 30 50 SEC63 40 Clone 1 Allele 1 = in-frame deletion of Y272-R273 (red) Allele 2 = in-frame deletion of G274-N300 (yellow + blue) Clone 2 Allele 1 = in-frame deletion of Y272-R273 (red) Allele 2 = in-frame deletion of G274-Q275 (yellow) Clone 1 Allele 1 = in-frame insertion C82 → VGI (yellow) Allele 2 = in-frame insertion (NNKIIIKIN) between N83 and R84 (arrowhead) Clone 2 Allele 1 = out-of-frame deletion starting at R75 (red) Allele 2 = out-of-frame deletion starting at E76 (blue) 399 390 380 360 370 350 340 330 310 320 300 290 230 240 250 260 270 280 220 200 210 170 190 180 150 160 140 130 100 110 120 90 80 70 60 50 40 SEC61A1 SEC62 20 30 10 ER-lumenal Cytoplasmic ER-lumenal Cytoplasmic bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary figure 2: Protein expression of the targeted SEC61/62/63 subunits in clonal mutant cell lines

A gRNA 2 WT A B C 50 SEC61A 37

1 2 B gRNA 1 gRNA 2 WT A B C D E F G H WT I J K L 15 15 SEC61B 10 10 75 75 HLA-GFP 50 50 HLA-I 37 37

1 2

C gRNA 1 gRNA 2 WT A B C D E F G H I 25 SEC61G 20 75 HLA-GFP 50 37 HLA-I

1 2 D gRNA 1 gRNA 2 WT A B C D E F WT G H I J

50 50 SEC62 37 37

1 2

E gRNA 1 WT A B C 150 100 SEC63 75 1 2 bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary figure 3: Amino acid and codon properties of the US2-, US11- and CD8 signal peptide 40

20 US2L

frequency per thousand codons 0 MNNLWKAWVGLWTSMGPLIR AUG AAC AAT CTC TGG AAA GCC TGG GTG GGT CTT TGG ACC TCC ATC GGT CCC TTG ATC CGC

20 US11L

frequency per thousand codons 0 MNLVMLILALWAPVAGS ATG AAC CTT GTA ATG CTT ATT CTA GCC CTC TGG GCC CCG GTC GCG GGT AGT

20 CD8L

frequency per thousand codons 0 MALPVSALLLPLALLLHAARP ATG GCC TTA CCA GTG ACC GCC TTG CTC CTG CCG CTG GCC TTG CTG CTC CAC GCC GCC AGG CCG

signal peptide sequence

hydrophobic residue polar residue charged residue rare codon cut-off rare codon bioRxiv preprint doi: https://doi.org/10.1101/653857; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary figure 4: The US2 signal peptide has weak signal peptide characteristics

1.0 0.8 0.6 US2L-US2

Score 0.4 C 0.2 NH 0.0 MNNLWKAWVGLWTSMGPLIRLPDGITKAGED 0 5 10 15 20 25 30 35

1.0 0.8 0.6 US2L-HA-US2

Score 0.4 HC 0.2 N 0.0 MNNLWKAWVGLWTSMGPL I RGGYPYDVPDYASALPDG I TKAGED 0 5 10 15 20 25 30 35 40 45

1.0 0.8 0.6 US11L-HA-US2

Score 0.4 NHC 0.2 0.0 MNLVMLILALWAPVAGSYPYDVPDYASALPDGITKAGED 0 5 10 15 20 25 30 35 40

1.0 0.8 0.6 CD8L-HA-US2

Score 0.4 NHC 0.2 0.0 MALPVTALLLPLALLLHAARPGGYPYDVPDYASALPDGI TKAGED cleavage probability 0 5 10 15 20 25 30 35 40 45 n-region probability h-region probability Position c-region probability