Genome
Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate- mediated intracellular calcium homeostasis
Journal: Genome
Manuscript ID gen-2019-0113.R2
Manuscript Type: Article
Date Submitted by the 11-Sep-2019 Author:
Complete List of Authors: Balasubramanian, Vidhya; Indian Institute of Technology Madras, Department of Biotechnology SRINIVASAN, BHARATH; Indian Institute of Technology Madras, DepartmentDraft of Biotechnology Intracellular calcium homeostasis, Golgi, IP3 receptor, Keyword: Drosophila melanogaster, Nucleobindin-1
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1 Title Page
2 Genetic analyses uncover pleiotropic compensatory roles for Drosophila 3 Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium 4 homeostasis 5
6 Authors: Vidhya Balasubramanian1 and Bharath Srinivasan1*
7 1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute 8 of Technology-Madras, Chennai, 600036, India. 9 Telephone: +9181222 48706 10
11 Email: [email protected]
12 *Corresponding Author
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27 Abstract:
28 Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly
29 localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative
30 involvements in intracellular calcium (Ca2+) homeostasis have however never been
31 phenotypically characterized in any model organism. We have analyzed an adult-viable mutant
32 that completely disrupts the G protein α-subunit Binding and Activating (GBA) motif of
33 Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious
34 fitness-related, morphological / developmental or behavioral abnormalities. A single copy of this
35 mutation or the knockdown of dmnucb1 in restricted sets of cells, however variously rescues
36 pleiotropic mutant phenotypes arising from impaired Inositol 1,4,5-trisphosphate receptor
37 (IP3R) activity (in turn depleting cytoplasmicDraft Ca2+ levels across diverse tissue types).
38 Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and
39 mobility improvements effected by IP3R mutants, in a Drosophila model of Amyotrophic
40 Lateral Sclerosis. Homology modeling-based analyses further predict a high degree of
41 conformational conservation in Drosophila, of biochemically validated structural determinants
42 in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of
43 NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed,
44 regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in
45 health and disease.
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47 List of Keywords
48 Nucleobindin-1, Intracellular calcium homeostasis, Golgi, IP3 receptor, Drosophila
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51 1. Introduction:
52 An elaborate ‘toolkit’ is well known to orchestrate Ca2+ signaling phenomena in diverse
53 vertebrate and invertebrate cell types with varied spatiotemporal dynamics, ranging from a brief
54 localized increase to repetitive spikes and waves spreading across wider regions. Ca2+ channels
55 on the plasma membrane and the IP3 receptor on the endoplasmic reticulum (ER) facilitate rapid
56 increases in cytosolic Ca2+. These are buffered by the action of various Ca2+-binding proteins
57 and also sequestered by Ca2+ pumps on the plasma membrane or intracellular membranes [such
58 as the Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA) or the Secretory Pathway
59 Ca2+/Mn2+-ATPase (SPCA), on the ER] and Ca2+ exchangers at both locations (Berridge et al.
60 2003; Chorna and Hasan 2012). The ER has traditionally been regarded as the primary store of
61 intracellular Ca2+ and its depletion is wellDraft established to activate the Store Operated Calcium
62 Entry (SOCE) pathway, to facilitate extracellular Ca2+ influx through the Orai plasma membrane
63 channel (Taylor and Machaca 2019). In recent years however, evidence has been accumulating
64 to suggest that other organelles such as the Golgi apparatus, mitochondria, peroxisomes and
65 endolysosomal compartments also store significant amount of ionic Ca2+, although the
66 functional implications of these stores are only beginning to be appreciated (Michelangeli et al.
67 2005).
68
69 Nucleobindin-1 (also known as NUCB1 or Calnuc) is a multi-domain EF-hand calcium-binding
70 protein, phylogenetically conserved from worms to humans (Aradhyam et al. 2010). It has been
71 identified as the major Golgi-associated calcium (Ca2+) binding protein and to be involved in the
72 establishment and maintenance of an agonist-mobilizable Golgi Ca2+ store, in a wide variety of
73 vertebrate cell and tissue types (Lin et al. 1998; Lin et al. 1999). Indeed, NUCB1 has also been
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74 identified as the second most abundant Golgi-associated protein in a quantitative proteomics
75 study evaluating fractions isolated from rat liver (Gilchrist et al. 2006).
76
77 Biochemical and cell culture-based approaches have made important contributions to the
78 understanding of select aspects of NUCB1 function (Garcia-Marcos et al. 2011; Kapoor et al.
79 2010; Weiss et al. 2001). Vertebrate NUCB1 has been shown through these approaches, to
80 harbor a motif of 15-25 amino acids (aa) that can directly bind to G protein α subunits and
81 stimulate their guanine nucleotide exchange activity, at the cytoplasmic surface of Golgi
82 membranes and without the involvement of a G protein coupled Receptor (GPCR) (Aznar et al.
83 2016; Garcia-Marcos et al. 2011). Among the small class of proteins currently known to contain
84 this G protein α-subunit Binding and ActivatingDraft (GBA) motif, vertebrate NUCB1 and its paralog
85 NUCB2 are the only members known to bind Ca2+ and the only ones localizing primarily to the
86 Golgi. Indeed, since the GBA motif in these proteins overlaps with one of their EF hands,
87 binding of Ca2+ has been shown to abolish their respective interactions with Gαi1/3 subunits.
88 Despite their relatively unique and highly interesting profiles, however, potential in vivo roles
89 for NUCB1 or NUCB2 have not been represented by informative mutant phenotypes in any
90 model genetic organism.
91
92 Drosophila melanogaster (henceforth referred to as, simply, Drosophila, except where it needs
93 to be distinguished from other Drosophila species) is among the most tractable experimental
94 organisms and routinely affords a wealth of resources for molecular genetic investigations,
95 especially in relation to Ca2+ signaling phenomena and the modeling of many human diseases
96 (Chorna and Hasan 2012; Ugur et al. 2016). NUCB1 is represented by a single copy gene in the
97 Drosophila genome, in turn encoding just a single isoform [as opposed to vertebrate genomes in
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98 which the NUCB1 and NUCB2 / NEFA genes both encode multiple isoforms (Kawano et al.
99 2000; Aradhyam et al. 2010)]. Potential hurdles arising from functional redundancy with respect
100 to in vivo genetic approaches are thus easily circumvented in Drosophila. Most of the functional
101 domains predicted for vertebrate NUCB1 are also conserved in the Drosophila homolog, which
102 shares an overall sequence similarity of 58% with its human counterpart (Otte et al. 1999).
103
104 This study was undertaken to examine whether genetic disruption of the GBA motif in the
105 Drosophila NUCB1 ortholog could be characterized in terms of discrete mutant phenotypes.
106 Our efforts were also aimed at clarifying whether such impairment could be compared or
107 genetically linked with independently documented phenotypes known to reflect specific
108 molecular aspects of intracellular Ca2+Draft homeostasis. Mutants corresponding to the Drosophila
109 IP3 receptor were preferred in this regard, since it represents genetically, the best-studied core
110 component of intracellular Ca2+ homeostasis in Drosophila (Chorna and Hasan 2012). Wherever
111 possible, knockdown approaches in restricted cell, tissue or organ types were also attempted to
112 supplement these primary goals. Our approaches were intentionally broad-based in design, as
113 opposed to being focused on a specific cell / tissue / organ type or biological process. This
114 aspect of our work was based not only on the wide expression patterns of NUCB1 in vertebrates
115 and invertebrates, but also on the fact that neither NUCB1, nor any GBA motif harboring protein
116 has been genetically analyzed in any model organism, to date.
117
118 We report that a piggyBac transposon insertion in the GBA motif of Drosophila NUCB1 (that
119 presumably renders the motif functionally inactive) does not affect the overall fitness of flies,
120 even in the homozygous condition. Flies carrying two copies of this insertion are also
121 phenotypically normal with regard to many different behavioral or morphological assessments.
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122 A single copy of this insertion however variously rescues wing posture, cold sensitivity and gut
123 immunity defects in multiple mutants deficient in IP3 levels or the expression / function of the
2+ 124 IP3 receptor (in turn, uniformly leading to depleted cytoplasmic Ca levels) (Ha et al. 2009;
125 Venkiteswaran and Hasan 2009). This mutation also appreciably augments the rescue of flight
126 ability and cold sensitivity defects in IP3 receptor mutants, by stocks carrying other mutations in
127 tandem, at the dSERCA and / or dOrai loci, respectively (Venkiteswaran and Hasan 2009). The
128 heterozygous GBA mutation additionally reverses lifespan and mobility improvements
129 independently wrought by single copies of two different IP3 receptor mutations, in flies
130 overexpressing a human gene implicated in Amyotrophic Lateral Sclerosis (Kim et al. 2012).
131 Knockdown of Drosophila nucb1 in specific subsets of cells and using two different RNAi lines,
132 phenocopies most of these results, albeitDraft to a lesser degree. Finally, homology modeling of the
133 GBA motif in Drosophila NUCB1 using its human counterpart as template, confirms the
134 structural conservation of many important residues required for potential binding of Gα
135 subunits, on both sides of the interaction. At the primary amino acid sequence level, the
136 conservation of multiple motifs and signals necessary for the localization of such interactions to
137 the cytoplasmic surface of Golgi membranes, are also seen to be conserved.
138
139 Results deriving from our unbiased genetic approach thus elucidate important modulatory
2+ 140 functions for Drosophila NUCB1 in intracellular IP3-mediated Ca homeostasis. These roles
141 may likely be associated with the Golgi and encompass diverse tissue types and biological
142 processes, including neurodegeneration in an established Drosophila model of human disease.
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144 2. Materials and methods:
145 Fly Genetics
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146 All Drosophila stocks were maintained on standard cornmeal agar food and raised at 25°C on a
147 12:12 hour light/dark cycle, unless otherwise indicated. The three itpr mutant alleles itprug3 /
148 TM6, Tb, itprwc361 / TM3, Tb and itprwc703 / TM3, Tb were kindly provided by Dr.Gaiti Hasan of
149 the National Center for Biological Sciences (NCBS), Bangalore. The dmnucb1c01508, dOrai11042
150 and Kum170 stocks were obtained from the Bloomington Stock Center and the two dmnucb1
151 RNAi lines from the National Institute of Genetics (NIG), Japan
152 (https://shigen.nig.ac.jp/fly/nigfly/). Additional information on various stocks used is listed in
153 Supplementary1 Table ST42.
154
155 Stocks carrying the dmnucb1c01508 transposon insertion (75A2 on chromosome 3L),
156 independently in tandem with each ofDraft three different point mutations in itpr (ug3, wc703 or
157 wc361 – all at cytological position 83A7-B1) were created by standard genetic recombination
158 crosses. A recombinant carrying dmnucb1 RNAi(3) recombined with itprug3 was also additionally
159 created, for use in specific experiments. Recombinants were screened on the basis of eye color
160 (scoring for the piggyBac insertion) and then lethality against each of the three itpr alleles,
161 followed by confirmation of both entities by DNA sequencing.
162
163 Revertants were identified on the basis of loss of eye color (scoring for excision of the piggyBac
164 insertion) induced by crosses with a piggyBac transposase stock and subsequently verified by
165 DNA sequencing. Revertants derived from the dmnucb1c01508 parent stock were designated as
166 dmnucb1c01508Rev1A, dmnucb1c01508Rev2A etc., while those derived from the recombinant stocks
167 outlined above were designated with the suffixes ‘Rev1R’, ‘Rev2R’ and so on. A total of five
1 Supplementary data are available through the journal web site, at: http://nrcjournalpress.com. 2 Supplementary Table ST4
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168 independent revertants each, was isolated for any given stock, and tested as required. The results
169 from only a maximum of two revertants are shown in each data figure to avoid further
170 overcrowding of the relevant graphs.
171
172 Choice of itpr (IP3 receptor) heteroallelic mutants
173 The itprsa54 mutant was not used in these and other assays to avoid possible complications
174 arising from crumpling of wing margins (Banerjee et al. 2004). For reasons unclear to us, the
175 itprka1091 mutation could not be recombined onto the dmnucb1c01508 chromosome, despite
176 repeated attempts. The itprwc703 / itprwc361 heteroallele has been shown to exhibit wild type levels
2+ 177 of IP3-stimulated Ca release from microsomal vesicles and was therefore not used in any of
178 our studies (Banerjee et al. 2006). Draft
179
180 Neurons cultured from itprug3/wc703 larvae are known to hypomorphically release significantly
2+ 181 smaller amounts of Ca from the ER store upon Inositol 1,4,5-trisphosphate (IP3) stimulation
182 and to exhibit reduced store-operated calcium entry through the plasma membrane, as well
183 (Venkiteswaran and Hasan 2009). Among the heteroallelic combinations that display fully
184 penetrant wing posture defects, itprug3/ wc361 has been recorded as the least severe, in terms of
185 flight impairments (Banerjee et al. 2004). While the missense substitution in itprug3 localizes to
186 the N-terminal ligand-binding domain of the fly IP3R and the mutation in itprwc703 alters a
187 conserved glycine residue in its modulatory domain, that in itprwc361 truncates IP3R by merely
188 15 residues at its C-terminal end, well beyond the Ca2+ channel domain (Joshi et al. 2004).
189 Based on these differential molecular and phenotypic criteria, itprug3/wc703 and itprug3/ wc361 were
190 used in all our experiments.
191
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192 RNA Isolation and RT-PCR
193 30 third instar larvae carrying either UAS-dmnucb1-IR insertion with tubulin-Gal4 were used
194 for RNA isolation, with wild type third instar larvae as control. Total RNA was isolated using
195 the Trizol method followed by cDNA synthesis for visualization on agarose gels. For semi-
196 quantitative RT-PCR, TaqMan reverse transcription reagents (Thermo Fisher Scientific,
197 Waltham, MA, USA, www.thermofisher.com) were used. Sequence information for primer
198 design is summarized in Supplementary Table ST53.
199
200 Inverse PCR
201 For verification of the piggyBac transposon insertions, the protocols outlined at
202 https://drosophila.med.harvard.edu/node/32609Draft (last accessed on June 4, 2019) were used, with
203 minor modifications and using primers specific for the PB or WH vectors, as required.
204
205 Fertility / Fecundity Assays
206 To determine the fertility of male or virgin female flies of a given genotype, individual flies of
207 either sex were single-pair mated in vials with freshly eclosed individual control (w1118) flies of
208 the opposite sex. After 7 days, the number of pairs that had produced any offspring was counted.
209 For the fecundity assays, single male–female pairs were placed into glass vials with 1 ml
210 unyeasted banana medium, which was replaced every 12 hours by a fresh vial containing banana
211 medium. The number of eggs laid was independently counted every 12 hours for 12 days, from
212 10 different vials for each genotype of interest, averaged and considered as the reproductive
213 output at age 12 days. Computation of SEMs, as well as t tests and plotting of the results were
214 all performed using GraphPad Prism 6.0 for Windows (GraphPad Software, La Jolla, CA,
3 Supplementary Table ST5
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215 U.S.A., www.graphpad.com) for these assays, as also the body weight, temperature-sensitive
216 paralysis and locomotor behavior assays, below.
217
218 Body weight
219 3 flies of any given genotype were placed in a 1.5 ml microfuge tube and a Sartorius precision
220 weighing balance was used to measure their combined weight, following the blanking of tube
221 weight.
222
223 Temperature-sensitive paralysis Assays
224 The original paralysis protocol as described in Sanyal et al., 2005, was modified, wherein a
225 water bath was used instead of the “SushiDraft cooker”. Paralysis was defined as the condition in
226 which the animal lies on its back with little or no movement of its legs and wings. All flies
227 tested were 1-2 days old (as a mixture of males and females). Test flies were exposed to the
228 restrictive temperature range of 39°C- 40°C, and paralysis profiles were obtained by introducing
229 batches of 10 flies at a time in glass vials, into the water bath. Percentages of immobile flies
230 were recorded from trials at 5.5 minutes for each trial, since 100% of Kum170 flies paralyze by
231 this time point, only to recover after 48 hours (Sanyal et al. 2005).
232
233 Wing Vein Patterning Analysis
234 Wings were dissected from adult flies, washed in 100% ethanol and mounted in Aquamount
235 mounting medium (Fisher Scientific, Hampton, NH, U.S.A., www.fishersci.com). To ensure that
236 handling or mounting did not affect wing polarity phenotypes, all wings were handled only in
237 the hinge region and both wild-type control and mutant wings were mounted under the same
238 coverslip, with weights added to flatten wing blades.
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239
240 Flight Assays
241 Flight tests were performed using the “cylinder drop” assay, as described in Banerjee et al. 2006.
242 A batch of 20 flies was tested in each trial and a minimum of five trials was carried out for each
243 genotype. Briefly, flies that dropped directly down the cylinder were collected in a vial kept on
244 ice underneath the lower opening of the cylinder and were scored as ‘flight defective’. Flies that
245 were able to hold onto the walls of the cylinder were considered as ‘fliers’. The fraction of flies
246 that dropped into the vial at the bottom of the cylinder scored against the total number of flies
247 tested determined the measure of flight defective organisms. t tests were performed to
248 statistically compare any two relevant genotypes.
249 Draft
250 Cold-sensitive lethality Assays
251 These experiments were performed mostly as outlined in Joshi et al. 2004. Timed and
252 synchronized egg collections were carried out for 6 hours at 25°C. Heteroallelic and
253 heterozygous larvae were identified based on the absence of the dominant Tubby phenotype
254 (marking TM6B) or the CyOGFP marker, as required, between 116-124 hours after egg laying
255 (AEL). Larvae were then transferred into vials of cornmeal medium lacking agar, grown at
256 17.5°C and screened for the number of survivors, at periodic intervals. Larval stages were
257 determined by the morphology of the anterior spiracles. For each time interval, a minimum of
258 100 larvae were screened in batches of 20 larvae each. One way ANOVA tests were performed
259 along with Dunnett’s multiple comparisons to determine statistical significance, using GraphPad
260 6.0 for Windows.
261
262 Survival Experiments
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263 For the yeast-based survival experiments (Fig. 4), flies of different genotypes were reared on
264 media composed of autoclaved cornmeal agar media supplemented with 2% heat-killed yeast
265 (‘dead yeast’ media) up to the early adult stage at day 2, as outlined in Ha et al. 2009. 2-day old
266 adult flies were then transferred into a fresh vial containing autoclaved cornmeal agar media
267 supplemented with 2% ‘live yeast’ or ‘dead yeast’ media. Dried live S. cerevisiae was purchased
268 from Lesaffre Yeast Corp. (Milwaukee, Wisconsin). Flies were then regularly transferred into
269 fresh vials with the requisite ‘live’ or ‘dead’ yeast media, every 4 or 5 days. For longevity /
270 lifespan assays, flies were maintained on regular cornmeal agar media and transferred to fresh
271 food every 4-5 days.
272
273 In both sets of experiments, flies were Draftmaintained at a density of 20 per vial and 5 independent
274 trials were carried out. The number of flies that survived was counted at periodic intervals,
275 wherein dead flies were scored by lack of responsiveness after tapping of the vials.
276
277 For the PLC β, as well as TDP-43 experiments, survival curves were compared using log-rank
278 analyses (Mantel–Cox test). In the former case, longevity curves were plotted by Kaplan–Meier
279 analysis for a more accurate representation. GraphPad Prism 6.0 for Windows was used in both
280 cases.
281
282 Locomotor behavior Assays
283 Climbing (negative geotaxis) assays were carried out using 14-day old adults of the same sex.
284 For each test run, 20 flies were placed in a standard 15 ml conical tube and tapped to the bottom
285 of the tube. Climbing indices were scored as the fraction of flies that reached the 10-ml mark
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286 after 5 seconds. A minimum of 5 trials for each genotype was performed and a total of 100 flies
287 (20 flies per batch) were screened.
288
289 Homology Modeling
290 Conformational modeling of protein side chains and loops was carried out using the ICM
291 package (Molsoft, San Diego, CA, U.S.A., www.molsoft.com), based on internal coordinate
292 definition of molecular objects, combined with computationally efficient Biased Probability
293 Monte Carlo (BPMC) optimization (Arnautova et al. 2011). The model was optimized in an
294 extended force field, including surface terms, electrostatics, and side chain entropy terms. The
295 quality of the resulting 3D model was assessed by a specialized ICM procedure called ‘Protein
296 Health’, which also predicts possible backboneDraft deviations between the homologues.
297
298 Web site references
299 Unless otherwise indicated, web sites corresponding to FlyBase, its sister sites (FlyAtlas, Flygut,
300 Berkeley Drosophila Genome Project) and the NIG RNAi stocks collection were all uniformly
301 last accessed just one week prior to the uploading of this manuscript, to verify the continued
302 authenticity of information cited.
303
304 3. Results:
305 The motif / domain-rich modularity of vertebrate NUCB1 is largely conserved in Drosophila
306 At least three novel sequence-specific features have been reported for vertebrate NUCB1 since
307 the publication of the only study to date (more than two decades back), directly dealing with the
308 characterization of its Drosophila counterpart (Garcia-Marcos et al. 2011; Nesselhut et al. 2001;
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309 Otte et al. 1999; Tsukumo et al. 2009). We therefore re-confirmed the nucleotide sequence of
310 the entire open reading frame of 1707 base pairs corresponding to the single copy Drosophila
311 nucb1 locus (henceforth CG32190 or dmnucb1), using multiple publicly available cDNA clones
312 (mainly IP03868 and IPO3768: http://www.fruitfly.org/DGC/index.html). dmnucb1 is annotated
313 on FlyBase4 (http://flybase.org/reports/FBgn0052190) to comprise a short exon followed by a
314 single intron and a large exon (Fig. 1A), in sharp contrast to the rat or human nucb1 genes,
315 which are thought to contain as many as 12 exons and 11 introns (Aradhyam et al. 2010). Our
316 sequencing efforts confirmed the prediction that dmnucb1 encodes a multi-domain polypeptide
317 (henceforth dmNUCB1) of 569aa, with an estimated molecular weight of 67.4kDa (Fig. 1B).
318 The predicted dmNUCB1 protein was verified to share extensive sequence similarity with both
319 human paralogs – Nucleobindin-1 (NUCB1Draft or Calnuc) and Nucleobindin-2 (NUCB2 or NEFA)
320 – particularly in the region between aa positions 57 and 345, where 48% of its residues are
4 The term ‘Nucleobindin 1’ or ‘NUCB1’ was used to represent the DNA-binding ability of this
protein, when it was first discovered (Miura et al. 1994). NUCB1 was later re-christened as
‘Calnuc’, to reflect its dual Ca2+ and DNA-binding functionalities and to distinguish it from the
NEFA / NUCB2 vertebrate paralog [which is thought to have been derived from Calnuc, long
after the segregation of a mammalian ancestor from an insect ancestor (Kawano et al. 2000; Lin
et al. 1998)]. Although both designations continue to be invoked in the primary literature,
throughout this manuscript, the terms dmnucb1 (in relation to the homologous single copy
Drosophila melanogaster locus or mutants thereof) and dmNUCB1 (to refer to the single
polypeptide it is thought to encode) are intentionally used, to maintain conformity with existing
Flybase nomenclature.
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321 identical and 66% are similar, in the case of NUCB1. dmNUCB1 also shares most of the
322 functional motifs and domains identified in vertebrate NUCB1 and NUCB2 (including, most
323 importantly, the GBA motif), in approximately the same relative positions. The Drosophila
324 homolog is however more than a 100aa longer than its vertebrate counterpart, features a
325 hypothetical third EF-hand motif and lacks the C-terminal leucine zipper, which is replaced
326 instead, by a Glutamine-rich repeat region that was originally termed (in the ‘pre-genome
327 sequencing’ era) as the ‘PVQ5’ repeat region (Otte et al. 1999). Detailed analyses of this repeat
328 sequence revealed that it is not fully conserved even in Drosophila simulans, the fly species that
329 is evolutionarily closest to Drosophila melanogaster, among those for which whole genome
330 sequencing information is publicly available (Clark et al. 2007). Further comparisons of
331 predicted sequences for this region Draft from Drosophila species that are more distant in
332 evolutionary terms (Drosophila virilis, the cactophilic, desert-inhabiting Drosophila mojavensis
333 or Drosophila Grimshawi, respectively) on the one hand or from the nematode, C.elegans, on
334 the other, led us to conclude that this region varies considerably and non-uniformly with respect
335 to overall length, number of ‘PVQ’ repeats (or even, quite simply, ‘Q’ repeats), as also
336 percentages of constituent amino acids, even within invertebrates [Appendix Fig. A2, Appendix
337 Table AT1]5. We have therefore taken the liberty of re-naming this portion of invertebrate
338 NUCB1 as simply, the ‘variable repeat region’ (Fig. 1B) [Appendix Figs. A1, A2].
5 A stand-alone ‘Appendix’ file catalogues the results of a more detailed bioinformatics-based
comparison study encompassing the hypervariable C-terminal region, all 3 EF hands, the Switch
II / α region in all isoforms of Gαi/o, the GBA motif, the Proline+2 signal, the N-terminal Golgi
Retention Sequence and the membrane lipid-anchoring motif from human and rat NUCB1 and
NUCB2, the C.elegans homolog and NUCB1 from 5 different Drosophila species.
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339
340 Ubiquitous knockdown of dmnucb1 causes early pupal stage lethality
341 RNAi lines carrying inverted repeat constructs corresponding to dmnucb1 on two different
342 chromosomes [NIG stocks 32190R-3: henceforth UAS-dmnucb1 RNAi(X) and 32190R-1:
343 henceforth UAS-dmnucb1 RNAi(3)] were chosen for use in some of our studies. Ubiquitous
344 transcriptional expression patterns for dmnucb1, as also for the translated dmNUCB1 protein,
345 during embryonic development, have already been documented (Otte et al. 1999). Further,
346 FlyAtlas documents the expression of dmnucb1 across all developmental stages, in a wide
347 spectrum of tissues (http://flyatlas.org/atlas.cgi?name=CG32190-RA). In keeping with these
348 observations, ubiquitous knockdown of dmnucb1 using the tubulin-Gal4 driver was found to
349 cause pupal lethality in both the RNAi Draftlines, although molting across all larval stages remained
350 unaffected (Supplementary Table ST1)6. It is assumed that the maternal component detected for
351 dmnucb1 in preblastoderm embryos, as also incomplete knockdown in both RNAi lines, are
352 together sufficient to ensure survival and proper development until the early pupal stage,
353 analogous to IP3 receptor gene (itpr) expression (Joshi et al. 2004; Otte et al. 1999). RT-PCR
354 analyses confirmed a significant knockdown of dmnucb1 transcript levels in both RNAi stocks
355 when driven by the tubulin-Gal4 driver (Fig. 1D) (Supplementary Fig. S1)7.
356
357 A mutant allele that disrupts EF-hand 2 and the contiguous GBA motif is adult viable and
358 exhibits no obvious abnormalities
359 While the aforementioned RNAi lines afforded the possibility of tissue-specific knockdown
360 strategies to examine dmnucb1 function, we further identified an interesting, publicly available
6 (Supplementary Table ST1) 7 (Supplementary Fig. S1)
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361 piggyBac transposon insertion mutant allele – PBac[PB]NUCB1[c01508] (henceforth
362 dmnucb1c01508) – for potential use in a variety of phenotypic assays. In conformity with data
363 documented on FlyBase (http://flybase.org/reports/FBti0040952.html), the c01508 insertion was
364 ascertained to localize to the middle of the second EF-hand (EF2) in dmnucb1 (GenBank
365 accession number CZ467221.1, Figs. 1B, C). The GBA motif of rat NUCB1 allows it to
366 exclusively and unconventionally interact with Gαi1 and Gαi3 on the cytoplasmic surface of
367 Golgi membranes (Garcia-Marcos et al. 2011) and was found to overlap with the loop region of
368 EF2. The c01508 insertion was confirmed to occur immediately prior to the predicted 7aa core
369 of this motif in dmnucb1, which overlaps with its EF2 motif (Figs. 1B, C). dmnucb1 levels
370 remained largely unaffected in the transposon insertion allele (Fig. 1D) (Supplementary Fig.
371 S1)8. Disruption of the GBA motif howeverDraft does not lead to lethality, infertility or any other
372 obvious fitness-related, morphological / developmental or behavioral abnormalities (as
373 evidenced from assays for fecundity, lifespan, body weight, molting profile, wing posture, wing
374 vein patterning, temperature-sensitive paralysis, flight or climbing ability, and multiple other
375 phenotypic traits), even in homozygotes (Supplementary Fig. S2)9. Given such a broad range of
376 phenotypic normalcy, the dmnucb1c01508 allele was considered a valuable genetic tool, since it
377 could allow us to examine the functional consequences of disrupting the dmNUCB1 GBA motif,
378 in sensitized genetic backgrounds already known to compromise canonical intracellular Ca2+
379 signaling. This allele was therefore preferentially used in all our experiments.
380
381 The dmnucb1c01508 heterozygous mutant rescues wing posture impairment and cold-sensitive
382 lethality in itpr mutants
8 (Supplementary Fig. S1) 9 (Supplementary Fig. S2)
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383 Viable heteroallelic mutant combinations that decrease cytoplasmic Ca2+ levels in varying
384 degrees have been extensively studied in terms of diverse mutant phenotypes (Chorna and Hasan
385 2012). Two such combinations – itprug3/wc703 and itprug3/wc361 (refer ‘Choice of itpr heteroallelic
386 mutants’ in ‘Materials and methods’, for more details) – afforded differently sensitized genetic
387 backgrounds to analyze whether modifier phenotypes could instead further refine our
388 characterization of the dmnucb1c01508 allele. A single copy of the c01508 insertion fully
389 suppressed the ‘spread out’ wing posture displayed by itprug3/wc703 and itprug3/wc361, such rescue
390 being partially phenocopied by restricted induction of dmnucb1 RNAi in aminergic neurons
391 (Banerjee et al. 2004) (Supplementary Table ST2)10. A single copy of dmnucb1c01508 was also
392 independently observed to greatly enhance the viability profile of both mutant heteroalleles at
393 572-580 hours AEL, when grown at 17.5Draft°C (Joshi et al. 2004) (Figs. 3A, B). In keeping with the
394 requirement for IP3R at the second instar larval stage, lethality suppression of both the itpr
395 heteroallelic combinations was seen to be less dramatic at 260-268 hours AEL (Supplementary
396 Figs. 6A, B)11. Since the Kum170 mutant can by itself dominantly effect considerable rescue of
397 cold-sensitive lethality in either itpr heteroallele, further introduction of the dmnucb1c01508 allele
398 predictably did not significantly alter viability profiles (Supplementary Figs. S5A, B)12.
399 Similarly, since the dmnucb1c01508 mutant more appreciably rescues cold-sensitive lethality in
400 itpr heteroalleles than Orai11042, only marginal improvements in viability profiles were seen
401 when both mutations were assayed together (Figs. 3A, B). Excision of the c01508 insertion
402 caused reversion of wing posture to the ‘spread out’ pattern, as also a pronounced reduction in
403 viability of both heteroallelic combinations, at colder temperatures (Figs. 2A; 3A, B). RNAi
10 (Supplementary Table ST2) 11 (Supplementary Figs. 6A, B) 12 (Supplementary Figs. S5A, B)
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404 induction was not attempted for the cold sensitivity assays, in view of the lower temperatures
405 involved.
406
407 Combined restoration of flight in itpr mutants by the dominant dOrai and dSERCA mutants is
408 further enhanced by the dmnucb1c01508 allele
409 Despite the rescue of wing posture defects, the overall percentage of flight abnormalities seen in
410 the itprug3/wc703 and itprug3/wc361 combinations in a “cylinder drop test” was reduced only to a
411 relatively minimal extent by the dmnucb1c01508 allele, even in recombinant genotypes carrying
412 two copies of the c01508 insertion (Supplementary Fig. S3)13. Further introduction of the
413 dominant Kum170 mutation also did not bring about a pronounced alleviation of these
414 impairments (Banerjee et al. 2006; SanyalDraft et al. 2005) (peach-colored 10th bar from the right in
415 Fig. 2B, compared with the dark green 4th bar from the right in Supplementary Fig. S4)14.
416 Significant suppression of flight impairment in either itpr heteroallele, by the heterozygous
417 dmnucb1c01508 mutant, was however observed against the genetic background of a single copy of
418 the dOrai11042 hypermorphic allele (Venkiteswaran and Hasan 2009). Further augmentation by
419 the dmnucb1c01508 homozygote was negligible (light olive green-colored 8th bar from the right
420 compared with the light pink-colored 7th bar in Supplementary Fig. S415; light pink-colored 11th
421 bar in Fig. 2B, as against the light olive green-colored 8th bar from the right in Supplementary
422 Fig. S4)16. Overall suppression was strongest in Kum170 / dOrai11042; itprug3-dmnucb1c01508 /
423 itprwc703-dmnucb1c01508 and Kum170 / dOrai11042; itprug3-dmnucb1c01508 / itprw361-dmnucb1c01508
424 quadruple mutants (dark brown 4th bar from the left and light olive green 1st bar from the left, in
13 (Supplementary Fig. S3) 14 (Supplementary Fig. S4) 15 Supplementary Fig. S4 16 (Supplementary Fig. S4)
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425 Fig. 2B). Recombinant stocks in which the c01508 transposon was excised (dmnucb1c01508Rev1R
426 and dmnucb1c01508Rev2R) restored flight ability levels to those seen in the Kum170 / dOrai11042;
427 itpru3 / itprw361 triple mutants (Fig. 2B). Knockdown of dmnucb1 in aminergic neurons
428 significantly recapitulated the augmentation of flight ability rescue in itpr mutants, by the
429 homozygous dmnucb1c01508 mutant in an Orai11042 mutant background (Fig. 2C).
430
431 Impairments in dmnucb1 expression or function ameliorates mortality phenotypes stemming
432 from compromised gut immunity
433 We also investigated the possible involvement of dmNUCB1 in a pathway that mobilizes
434 intracellular Ca2+ from the ER, without the genetic background of itpr mutations or knockdown,
435 but by attenuating instead, the upstreamDraft production of IP3. The Gαq-PhospholipaseC-β (PLCβ )-
436 Ca2+-Dual Oxidase (DUOX) pathway in Drosophila is known to dynamically regulate host
437 immune response and associated organismal survival by controlling the propagation of essential
438 food-borne nutritional microbes in gut epithelia (Ha et al. 2009). As reported earlier, gut-specific
439 knockdown of PLCβ (using the NP3084-Gal4 driver) significantly lowered survival rates in fly
440 populations raised on live yeast media, when compared with conditions in which heat-killed
441 yeast supplements were used instead (Figs. 4A-D) (Supplementary Fig.S7)17. Against the
442 background of PLCβ knockdown, introduction of a single copy of the dmnucb1c01508 allele or co-
443 expression of UAS-dmnucb1 RNAi in the gut, however noticeably increased alike, the overall
444 fraction of flies that survived live yeast treatments (Figs. 4A, C, D). Pertinently, Flygut records
445 appreciable expression of dmnucb1 in the crop, cardia / R1, R2 and R5 regions of the gut:
446 https://flygut.epfl.ch/expressions/show_by_gene?flybase_gene_id=FBgn0052190.
447
17 (Supplementary Fig. S7)
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448 Once again, excision of the c01508 insertion effected reversion of survival patterns on live yeast
449 media, in a manner comparable to those seen in the NP3084-Gal4 > UAS-PLCβ RNAi
450 background, alone (dmnucb1c01508Rev1A, dmnucb1c01508Rev2A: Fig. 4A). Control dmnucb1c01508
451 stocks (homozygotes or heterozygotes), or the two different RNAi lines driven by NP3084-Gal4
452 alone, did not exhibit any abnormalities in these assays. All stocks employed were also found to
453 be phenotypically normal on ‘dead yeast’ media (Supplementary Fig. S7)18.
454
455 We additionally observed that a control RNAi stock corresponding to the CG31650 locus (also
456 predicted to harbor two EF-hand motifs, but not a GBA motif and annotated on FlyBase as
457 localizing to the ER and not the Golgi: http://flybase.org/reports/FBgn0031673), did not display
458 any modifier phenotypes, in similar Draft assays (Supplementary Fig. S8A)19. This experiment
459 allowed us to infer that the rescue effects seen in the case of the dmnucb1 RNAi lines are
460 relatively specific and also not the result of background competition for Gal4 protein in
461 genotypes carrying two different UAS constructs.
462
463 The neuroprotective effects of itpr mutations in human TDP-43 overexpressing flies are reversed
464 by the dmnucb1c01508 allele or dmnucb1knockdown in tandem
465 Finally, we examined dmnucb1-itpr genetic interactions within the context of a recognized
466 Drosophila model of Amyotrophic Lateral Sclerosis. It has been reported that three different
467 mutant alleles of itpr can dominantly rescue mobility and lifespan defects in flies that
468 transgenically overexpress the human 43kDa TAR DNA-binding domain protein (TDP-43),
2+ 469 implicating IP3-gated cytoplasmic Ca as a key regulator of TDP-43 induced neurodegeneration
18 (Supplementary Fig. S7) 19 (Supplementary Fig. S8A)
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470 (Kim et al. 2012). We found that when UAS-dmnucb1RNAi was co-expressed in motor neurons,
471 improvements effected by two different itpr mutations (ug3 and 90B.0, respectively) in the
472 climbing index of TDP-43 expressing flies, were considerably reversed (Fig. 5A). These results
473 were comparably phenocopied by recombinants carrying a single copy each of dmnucb1c01508,
474 with either the itprug3 or the itpr90B.0 alleles (Fig. 5A). Similarly, the itprug3-dmnucb1c01508
475 recombinant displayed a pronounced reversion of mean lifespan gains brought about by the
476 itprug3 mutation alone, in flies overexpressing human TDP-43. This diminished viability profile
477 was also independently observed when dmnucb1 RNAi was co-expressed in motor neurons (Fig.
478 5B, C). Climbing and lifespan profiles in excision stocks lacking the c01508 insertion were
479 further observed to be comparable to those seen in the itprug3 or 90B.0 / D42-Gal4 > UAS-TDP-43
480 parent stocks (Fig. 5A, B). Draft
481
482 Once again, control experiments with the CG31650 RNAi stock did not recapitulate in both, the
483 locomotion, as well as the survival assays, the modifier phenotypes seen in the case of the
484 dmnucb1 RNAi lines, thus attesting to the specificity of our results (Supplementary Figs. S9A,
485 B)20. Since UAS-TDP43 is the only overexpression stock used in our studies, an additional
486 control experiment using a Dicer expressing UAS line, in the absence of any RNAi lines,
487 independently also further ruled out background competition effects for the Gal4 protein as a
488 putative experimental basis, for results observed with the dmnucb1 RNAi lines (Supplementary
489 Fig. S9C, D)21.
490
20 (Supplementary Figs. S9 A, B) 21 (Supplementary Figs. S9 C, D)
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491 Homology modeling reveals all crucial residues required for the potential Golgi-associated
492 interaction of dmNUCB1 with G protein α subunits to be appreciably conserved
493 The predicted sequence of the extended GBA motif in dmNUCB1 was homology modeled
494 against the 3D coordinates of human NUCB1’s putative Gαi-binding sequence, in turn threaded
495 over the structural coordinates of the synthetic peptide KB-752 complexed with human Gαi1 (de
496 Alba and Tjandra 2004; Johnston et al. 2005). Human and Drosophila dmNUCB1 alike, form a
497 helix with KB-752, in which the side chains of hydrophobic residues corresponding to positions
498 3, 6 and 7 from the core GBA consensus sequence of each entity very similarly dock onto the
499 α3/ Switch II hydrophobic cleft of the Gαi subunit (Garcia-Marcos et al. 2011) (Figs. 6A, B).
500 The positively charged K248 residue which has been shown in vertebrates to be important for
501 electrostatic interaction with the negativelyDraft charged E314 residue is also conformationally
502 conserved in dmNUCB1, wherein the E314 counterpart is represented by a negatively charged
503 D310 substitution (Fig. 6C) [Appendix Figs. A4(a), (b)].
504
505 Multiple other determinants hypothesized to be important either for NUCB1-Gαi interactions or
506 for its localization to the Golgi in vertebrates (the Proline+2 ER export / Golgi transport signal in
507 dmNUCB1 – 2aa away from the signal peptide cleavage site, the Leucine-Isoleucine-rich N-
508 terminal Golgi Retention Sequence in dmNUCB1 and the myristoylation / palmitoylation-based
509 motif for membrane lipid anchoring of Gα subunits) are also seen to be conserved at the
510 predicted primary amino acid sequence level in Drosophila melanogaster [Appendix Figs. A3-
511 7]. All of the sequence determinants outlined above for Gαi are surprisingly also found to be
512 conserved in both isoforms of Gαo in Drosophila melanogaster (unlike in vertebrates, where
513 preferential binding of Gαi1/3 by NUCB1 binding over Gαo subunits is determined by specific
514 residues). More importantly, all the residues critical for potential Gαi/o binding by NUCB1 on
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515 the one hand or all the motifs and signals necessary for their interaction-specific subcellular
516 localization on the other, are found to be conserved in protein sequences predicted for NUCB1
517 homologs from 4 other Drosophila species (Drosophila simulans, Drosophila mojavensis,
518 Drosophila virilis and Drosophila grimshawi), as well as the nematode, C.elegans. Such
519 conservation is seen regardless of their variable habitats or evolutionary distances from
520 Drosophila melanogaster [catalogued in great detail in Appendix Figs. A3-7]. This is in sharp
521 contrast to the considerable variation seen with regard to the C-terminal repeat region, in these 6
522 different invertebrate species [Appendix Figs. A1, A2, Table AT1].
523
524 Independently, the c01508 insertion was observed to introduce 3 in-frame stop codons in the
525 corresponding mutant allele, which is thereforeDraft predicted to result in a truncated NUCB1 protein
526 lacking the GBA motif and EF2, as also the variable repeat region (Fig. 6D).
527
528 4. Discussion:
529 Conventional trimeric G protein-based signaling is typically achieved through the activation of
530 Gα subunits by an extracellular ligand-bound GPCR at the plasma membrane, in its capacity as a
531 guanine nucleotide exchange factor (GEF). To the best of our knowledge, the work presented
532 here represents the first set of functionally-linked in vivo phenotypes, in any organism,
533 correlating primarily with a disruption of the GBA motif in any nonreceptor GEF. Further, of
534 the limited set of 5-6 proteins currently known to harbor a GBA motif, NUCB1, and its
535 vertebrate paralog, NUCB2, are the only ones whose activation of Gα subunits is regulated by
536 levels of intracellular Ca2+, and that too, at the level of an intracellular, organellar membrane
537 (Aznar et al. 2016; Garcia-Marcos et al. 2011). Our results thus acquire added significance in
538 light of the compensatory roles documented for dmNUCB1 in intracellular Ca2+ homeostasis,
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539 across diverse cell and tissue types, as also its association with the Golgi in insects and
540 vertebrates, alike (Kawano et al. 2000; Otte et al. 1999).
541
542 A single copy of the c01508 transposon insertion is sufficient to genetically modify six different
543 IP3-mediated functional contexts and corresponding mutant phenotypes. The rescue of wing
544 posture and cold sensitivity defects in itpr mutants is reminiscent of similar results reported for
545 the Kum170, and independently, the dOrai11042 mutant alleles (Banerjee et al. 2006;
546 Venkiteswaran and Hasan 2009). The former has been shown to delay cytoplasmic Ca2+
547 clearance following neuronal depolarization, and the latter, to function as a hypermorph in
548 facilitating extracellular Ca2+ entry, via the SOCE pathway (Sanyal et al. 2005; Venkiteswaran
549 and Hasan 2009). A single copy ofDraft the itprug3 mutation has conversely been reported to
550 dominantly suppress wing posture phenotypes in wavy (corresponding to fly IP3 Kinase 2)
2+ 551 mutants by reducing the availability of IP3 and therefore cytoplasmic Ca (Dean et al. 2016).
552 Independently, the improvements in lifespan and mobility by dominant itpr mutations, of flies
553 overexpressing human TDP-43 that we have reproduced in our studies, have been previously
2+ 554 linked in vivo to IP3-gated Ca release (Dean et al. 2016; Kim et al. 2012). Null mutations in the
555 fly ortholog of TDP-43, on the other hand, have very recently been shown to exhibit severe
556 locomotion defects in larvae, that are mediated by a reduction in the expression of a type II
557 voltage-gated calcium channel, cacophony (cac) (Lembke et al. 2019). Finally, dDUOX
558 overexpressing flies have been observed to exhibit a ‘held out’ wings phenotype, similar to that
559 seen in the itpr heteroalleles in our study (Anh et al. 2011). In vivo photobleaching FRET
2+ 560 analyses have also been independently used to demonstrate the integral role of IP3-gated Ca in
561 modulating dDUOX activity towards gut immunity in Drosophila (Ha et al. 2009). It thus
562 appears reasonable to speculate that enhanced availability of cytoplasmic Ca2+ may be a
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563 common factor underlying all the dmnucb1 associated modifier phenotypes we have observed,
564 against the genetic background of Drosophila itpr mutants.
565
566 The Kum170and dOrai11042 alleles are however, homozygous lethals and like other fly mutants
567 that reflect an unregulated excess of cytoplasmic Ca2+, display discrete mutant phenotypes, in
568 and of themselves (Ha et al. 2009; Lembke et al. 2019; Sanyal et al. 2005; Venkiteswaran and
569 Hasan 2009). The dmnucb1c01508 allele, in sharp contrast, is homozygous viable and
570 phenotypically normal with regard to more than 12 broad phenotypic traits assayed, despite the
571 fact that dmnucb1 is as widely expressed as the Orai channel or the SERCA pump in Drosophila
572 and that both EF-hand 2 (EF2) and the GBA motif are almost completely disrupted in this
573 mutant. These observations in turn appearDraft to suggest that the mutation in dmnucb1c01508 causes a
574 regulated enhancement in the cytoplasmic Ca2+ pool which may be enough to modify mutant
2+ 575 phenotypes in genetic backgrounds characterized by the lack of IP3-mediated Ca , but not
576 enough to cause lethality or to manifest other mutant phenotypes, by itself. Indeed, tissue-
577 specific knockdown of dmnucb1 phenocopies many of the results obtained with the
578 dmnucb1c01508 mutant, but often to a lesser degree, while ubiquitous knockdown of dmnucb1
579 causes pupal lethality (Supplementary Table ST1)22. Furthermore, the transposon insertion in
580 dmnucb1c01508 likely truncates the dmNUCB1 protein, resulting in complete deletion of the GBA
581 motif core and more than 250 residues downstream of it (Fig. 6D). A single copy of this
582 insertion is nonetheless enough to pleiotropically rescue diverse phenotypes uniformly linked to
2+ 583 IP3-mediated Ca homeostasis (Figs. 2-5). A more detailed evaluation of our findings is thus
584 called for on multiple levels, to hypothesize on the possible subcellular source and mechanism
585 underlying the regulated mobilization of intracellular Ca2+ in the dmnucb1c01508 mutant.
22 (Supplementary Table ST1)
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586
587 Mammalian dmNUCB1 has been recognized as an unusual protein in localizing to the Golgi
588 lumen, while also belonging to a soluble cytosolic pool (Lin et al. 1998; Lin et al. 1999; Weiss
589 et al. 2001). Drosophila NUCB1 has been localized in Schneider tissue culture cells to small
590 cytoplasmic clusters, very likely the Golgi apparatus, while the Spodoptera frugiperda homolog
591 has been localized in electron microscopic studies to the cis-Golgi cisternae and networks, in a
592 pattern that closely parallels that of mammalian cells (Kawano et al. 2000; Otte et al. 1999). At
593 the amino acid sequence level we additionally find that the Proline+2 ER export signal, as also
594 the Leucine-Isoleucine rich Golgi Retention Sequence (GRS), are both conserved across 5
595 different Drosophila species, regardless of their diverse habitats (Nesselhut et al. 2001;
596 Tsukumo et al. 2009) [Appendix Figs.Draft A5, A6]. Furthermore, analogues of eight different
597 residues that have been highlighted as key determinants of the GBA-mediated interaction of rat
598 NUCB1 with Gαi1/3 are also found to be conserved in these same 5 Drosophila species,
599 including appreciable structural conservation in Drosophila melanogaster (Garcia-Marcos et al.
600 2011) (Figs. 6A-C) [Appendix Figs. A4A(a), (b), Appendix Table AT2]. Pertinently, the GBA
601 motif of GBAS-1 – a nonreceptor GEF found only in worms – has been demonstrated to activate
602 mammalian Gα subunits, thereby substantiating the notion that these modules are functionally
603 preserved across evolutionarily highly divergent proteins, but very sensitive to even subtle
604 single aa variations (Coleman et al. 2016). Finally, consensus N-terminal myristoylation and
605 palmitoylation sequences that are known to facilitate anchoring of Gαi1/3 to the cytoplasmic
606 face of Golgi membranes in vertebrates are also fully conserved in the lone dGαi isoform in
607 these 5 Drosophila species (Weiss et al. 2001) [Appendix Fig. A7]. It is thus highly plausible
608 that the dmNUCB1-Gαi partnership is functionally conserved in Drosophila, at an analogous
609 subcellular location. The stringent preference by mammalian dmNUCB1 for Gαi1/3 over Gαo
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610 subunits may however have been acquired after the segregation of vertebrate dmNUCB1 from
611 an invertebrate ancestor [Appendix Fig. A7].
612
613 EF1 has been postulated in vertebrates as the main Ca2+-binding site, with EF2 showing
614 relatively limited affinity for Ca2+ ions (Lin et al. 1999). Mere disruption of EF2 would in any
615 case, likely be insufficient to account for most of our results, given the amounts of Ca2+ ions
616 normally released by IP3-gated release from the ER store, in Drosophila (Venkiteswaran and
617 Hasan 2009). EF2 is also found to be less stringently conserved across evolution and other
618 regions in dmNUCB1 may hypothetically be invoked in a functionally substitutive capacity (Lin
619 et al. 1999; Miura et al. 1994; Otte et al. 1999; Zhou et al. 2006) [Appendix Figs. 3(a)-(c)].
620 These factors may also concurrently helpDraft explain why the phenotypes documented here for the
621 dmnucb1c01508 allele manifest only in the sensitized background of heteroallelic itpr mutants
622 and other similar genetically compromised contexts, despite the inactivation of EF2. The Ca2+-
623 enrichment in the Golgi is known to exceed 0.1mM in vertebrates and rat NUCB1 has been
624 shown to be abundant to the tune of 0.38% of the total Golgi protein content (3.8μg NUCB1/mg
625 Golgi protein, 2.5 NUCB1 molecules/rat kidney cell) (Lin et al. 1999). Overexpression of
626 NUCB1 has also been reported to increase agonist and thapsigargin releasable Ca2+ from the
627 Golgi store in HeLa cells (Lin et al. 1999). If similar criteria are found to hold true in
628 Drosophila as well, the GBA motif thus comes into sharper focus, as also the Golgi Ca2+ store.
629 Dosage compensation is unlikely to play a primary role in the modifier phenotypes we observe,
630 since homozygous and heterozygous dmnucb1c01508 are not significantly different from each
631 other in terms of the degree of rescue effected in 4 of the 6 assays employed and since many of
632 our results are also phenocopied by RNAi-based knockdown of dmnucb1. The C-terminal repeat
633 region is also likely irrelevant to our findings, since it is seen to vary considerably in multiple
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634 aspects, even among different species of invertebrates and is likely truncated in the
635 dmnucb1c01508 mutant (Fig. 6D) [Appendix Fig. A1]
636
637 The Golgi-localized physical interaction of dmNUCB1 with dGαi/o, as also the premise that
638 dmNUCB1c01508 encodes a truncated protein remain to be more directly substantiated at – for
639 example – the levels of antibody-based biochemical characterization and immunofluorescence
640 microscopy. Pending such verification, in light of the homology modeling data we have
641 reported and other associated observations outlined above, we tentatively predict that the
642 truncated dmNUCB1c01508 protein can likely no longer functionally partner with Golgi
643 membrane-anchored dGαi/o in flies. Our speculations also tend towards favoring the intuitively
644 appealing notion that the loss of this interactionDraft in turn leads in this mutant, to release of Ca2+
645 from the Golgi luminal store (or alternatively, an attenuated capacity to replenish it) or Ca2+
646 microdomains along the cytoplasmic surface of the Golgi. These eventual consequences may
647 plausibly be facilitated through intermediate effectors, even if IP3R (or the SERCA pump), for
648 example, may not be the immediate downstream targets of dmNUCB1 mediated dGαi/o
649 signaling.
650
651 Initial support in favor of these hypotheses comes from two sets of genetic experiments
652 involving solely, the gut immunity assays (since this functional context lends itself most easily
653 to genetic manipulation). Co-expression of the Golgi-specific isoform of the Drosophila
654 homolog (SPoCK-AGolgi) of the secretory pathway Ca2+ /Mn2+-ATPase (SPCA) family of
655 vertebrate Ca2+ pumps, with PLCβ RNAi in the gut, was seen to reverse the rescue of lifespan
656 reduction on live yeast media, as effected by a single copy of dmnucb1c01508 (Southall et al.
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657 2006) (Supplementary Fig. S10A)23. It is reasonably presumed that the increase in cytoplasmic
658 Ca2+ wrought by dmnucb1c01508 is countered herein, via the pumping back of Ca2+ into the Golgi
659 by SPoCK-AGolgi. Independently, whereas co-expression of a truncated version of dmnucb1
660 (lacking the coding region for the entire GBA motif and all amino acids beyond), with PLCβ
661 RNAi in the gut, failed to reverse lifespan rescue by the dmnucb1c01508 allele on live yeast media,
662 co-expression of full-length dmnucb1 successfully reverted the viability profile to levels
663 comparable to those seen in the case of NP3084-Gal4 > UAS-PLCβ RNAi (Supplementary Fig.
664 S10B)24. Once again, it is assumed in these complementation assays, that the functional GBA
665 motif in full-length dmnucb1 suppresses the elevation of cytoplasmic Ca2+ levels caused by a
666 single copy of the dmnucb1c01508 allele by titrating or competing out the effects of the truncated
667 dmNUCB1c01508 protein. These experimentsDraft offer preliminary evidence that dmNUCB1 may be
668 exerting its effects in a functionally and/or dominantly epistatic, Golgi-associated capacity, in
669 relation to IP3 receptor function. Preliminary investigations also reveal that overexpression of a
670 constitutively active form of dGαi (UAS-dGαiQ205L) can play a role similar to overexpressed
671 full-length dmnucb1 in overcoming the modifier effects of the dmnucb1c01508 allele, in gut
672 immunity assays (Ogden et al. 2008). Since the putative involvement of two different isoforms
673 of dGαo can potentially complicate this whole functional context in Drosophila, these
674 possibilities are being exhaustively addressed through the use of constructs carrying
675 constitutively active versions of these isoforms (dGαoaQ205L and dGαobQ205L, respectively),
676 as well.
677
23 (Supplementary Fig. S10A) 24 (Supplementary Fig. S10B)
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678 The fact that a single copy of the c01508 insertion can by itself considerably modify phenotypes
679 observed across varied assays, and in a manner not significantly different from corresponding
680 effects seen in the case of the dmnucb1c01508 homozygote, deserves further discussion. It has
681 been postulated that in resting conditions, the cytosolic concentration of free Ca2+ in rat tissues
682 (50-100 nM) is significantly lower than the Kd value of NUCB1 for Ca2+ binding (~7μM), thus
683 enabling its activation of rat Gαi1/3 (Garcia-Marcos et al. 2011). Similar considerations may be
684 extrapolated in Drosophila, based on in vivo Ca2+ measurements from primary neuronal cultures
685 (Venkiteswaran and Hasan 2009). The Ca2+-bound conformation in vertebrate NUCB1 is
686 thought to bring into play key intramolecular contacts in rat NUCB1 and subsequent
687 conformational changes that effectively preclude Gαi1/3 linkage (Garcia-Marcos et al. 2011).
688 These molecular criteria may hypotheticallyDraft also be invoked in Drosophila, at the levels of
689 primary or secondary structure (Figs. 6A-C) [Appendix Figs. A3, A4(a), 4(b), Table AT2]. In
690 animals carrying a single copy of the dmnucb1c01508 insertion, we hence additionally speculate
691 that some of the Ca2+ from the initial release may bind to the wild type dmNUCB1 copy,
692 competitively displacing dGαi/o subunits in the process, and in turn stimulating additional
693 rounds of cytosolic Ca2+ enhancement. An amplification loop is thence presumed in our
694 hypothesis, to progressively cause sufficient build-up of cytosolic Ca2+ levels, thus rationalizing
695 the single-copy rescue of most of the itpr mutant phenotypes analyzed here. Knockdown
696 phenotypes may also be similarly rationalized, albeit with less dramatic impact. In the genetic
697 background of mutant IP3Rs however, Ca2+ release may plausibly occur through the Ryanodine
698 receptor, SERCAs yet undiscovered Golgi-localizing entities (such as two-pore channels or
699 members of the TRP superfamily of channels) or by increasing the quanta of Ca2+ release
700 (Venkiteswaran and Hasan 2009).
701
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702 More convincing support for our hypotheses can only come from in vivo Ca2+ measurements
703 involving these same stocks or more informatively, transgenics carrying point mutations in
704 important residues of the GBA motif (especially ones not equating with canonical residues in the
705 EF2 loop) and/or the Pro+2 signal, for example. Our own experiments thus only represent initial
706 forays towards elucidating the functional roles speculated upon for dmNUCB1. It must also be
707 added that although haploinsufficiency-based explanations cannot be fully excluded, it is
708 unlikely that they can sufficiently account for many of our results, in as much as the
709 dmnucb1c01508 allele is phenotypically normal with regard to a wide variety of assessments.
710 Since our analyses predict the dmNUCB1-dGαi interaction to occur on the cytoplasmic surface
711 of the Golgi, our hypotheses also do not factor in a role for the luminal pool of dmNUCB1,
712 which remains to be explored, by other Draftmethodologies.
713
714 Broadly speaking, the results communicated here help lay the foundation for a more thorough
715 dissection of the mechanisms by which dmNUCB1 can pleiotropically compensate for defects
2+ 716 arising from IP3-mediated Ca homeostasis, across diverse biological processes and cell types.
717 Our hypotheses are perhaps best validated within the context of the complex Drosophila flight
718 circuit which lends itself to a whole gamut of genetic manipulation strategies, while combining
719 physiological recordings at flight neuromuscular junctions with highly informative Ca2+ imaging
720 approaches (Venkiteswaran and Hasan 2009). These approaches have thus helped elucidate that
721 Ca2+ release through ITPR and the SOCE pathway jointly function as two different components
722 to enable normal flight patterns (Hasan and Venkiteswaran 2010). Our own efforts reveal that
723 the dmnucb1c01508 allele – like the Kum170 mutant, but unlike the Orai11042 mutant – can rescue
724 wing posture but not flight defects in itpr mutants, although it can further augment flight rescue
725 in Kum170 / Orai11042 double mutants. These observations raise the intriguing possibility that the
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726 putative Golgi store derived contribution may constitute a third component of the overall Ca2+
727 signal in orchestrating normal flight, beyond functionally substituting for either of these two
728 core components, in a merely restorative capacity (Fig. 2B) (Supplementary Fig. S4)25. Such an
729 additional component may contribute to the modulation of the intensity, amplitude and / or
730 frequency of an integrated or summated signal. Its participation would also imply that the
731 subcellular origin and nature of Ca2+ signals may be more deterministic for specific rescue
732 phenotypes than mere compensation of reduced cytosolic Ca2+ levels by subcellular sources
733 extraneous to a well-defined behavioral circuit. Other aspects of our work also indicate however
734 that dmNUCB1 (and by extension, the Golgi Ca2+ store) and Kum / dSERCA may have greater
735 functional roles to play in determining the overall cold-sensitive viability response, than dmOrai
736 (and the SOCE pathway). Draft
737
738 Although we ourselves have only explored compensatory roles for dmNUCB1, localized Ca2+
739 transients or plumes are well known to activate Ca2+ dependent signalling molecules already
740 located on or around the Golgi in vertebrate cells, such as Calpain or NCS-1, or to recruit
741 inactive signaling molecules such as Hippocalcin and RasGRP1 to Golgi membranes, thereby
742 activating them (Dolman and Tepikin 2006). These microdomain building blocks can also
743 combine to produce larger microdomains in modulating these events at such localized
744 subcellular regions (Berridge 2006). Organellar membrane-specific G protein involvements can
745 be envisioned as an efficient means to more tightly regulate these localized Ca2+ fluxes,
746 especially in neurons, where both pre- and post-synaptic events are controlled by miniaturized
747 Ca2+ signaling phenomena (Berridge 2006). The fact that Gα subunit activation by NUCB1 can
748 itself be regulated by levels of Ca2+ localizing to the cytoplasmic surface of an unconventional
25 (Supplementary Fig. S4)
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749 intracellular store adds an attractive dimension to these speculative considerations. Approaches
750 combining the imaging power of newly available Golgi-specific in vivo Ca2+ sensors in flies,
751 with the reliability of well-established electrophysiological techniques, may conceivably be
752 more useful in elucidating potential roles, if any, for dmNUCB1 in GBA-mediated Ca2+
753 microdomain signaling (Alonso et al. 2017). The dmnucb1c01508 mutant likely offers a uniquely
754 valuable tool for such explorations.
755
756 Centrins are the only other class of Ca2+-binding proteins known to interact with Gα subunits,
757 but lack the GBA motif and differ from NUCB1 in other fundamental aspects as well (Trojan et
758 al. 2008) (Supplementary Table ST3)26. On a related note, Cab45 is the only other Golgi-
759 associated Ca2+-binding protein known,Draft to date, but also lacks a GBA motif (Scherer et al.
760 1996). Recently, a GBA motif containing PLCδ-4b protein has been discovered, but shown to
761 function more as a G protein regulator, than a PLC (Maziarz et al. 2018). Our in vivo genetic
762 approaches may thus help lay the broad groundwork for investigating dmNUCB1 as a unique
763 convergence point possibly integrating cytoplasmic Ca2+ signaling with G protein-mediated
764 regulation of the Golgi Ca2+ store on the one hand and the ER store / SOCE pathway, on the
765 other. The combined twin capabilities of Ca2+ binding and Gα subunit interaction, in other
766 words, may afford dmNUCB1 a uniquely exquisite versatility as a molecular rheostat (as has
767 already been proposed for the GBA motif containing GIV/Girdin in a qualitatively different
768 capacity) in homeostatically linking otherwise compartmentalized aspects of intracellular
769 calcium signaling (Ghosh et al. 2011; Trojan et al. 2008) (Supplementary Table ST3)27.
770
26 (Supplementary Table ST3) 27 (Supplementary Table ST3)
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771 Finally, although our work has been restricted to the putative involvement of dmNUCB1 in
772 ALS, the tools we have employed may also help delineate fundamental aspects of the etiologies
773 of other related human diseases, in which Golgi-modulated cytoplasmic Ca2+ levels have been
774 implicated (Li et al. 2013). Indeed, preliminary results from some of our collaborative efforts
775 also indicate that the elevation of cytosolic Ca2+ levels by a single copy of the dmnucb1c01508
776 mutant may be linked to Drosophila models of α-Synuclein toxicity, in a manner that counters
777 the effects of the SPoCKGolgi isoform (Buttner et al. 2013). The GIV-Gαi binding interface has
778 also recently been validated as a “druggable” protein-protein interaction, negating earlier
779 skepticism to the contrary (DiGiacomo et al. 2017). Extrapolating similar criteria to the NUCB1-
780 Gαi interface, the resources analyzed in our studies – if validated more extensively – may
781 potentially also help lay the foundationsDraft for novel avenues of similarly targeted therapeutic
782 intervention.
783
784 Competing interests
785 The authors declare that they have no competing interests.
786
787 Author contributions
788 V.B. and B.Sr. together designed all the experiments. B.Sr. supervised the entire study and V.B.
789 performed all the experiments. V.B. and B.Sr. jointly analyzed all the results, as well as wrote
790 the manuscript. Both authors have read and approved the final manuscript.
791
792 Acknowledgements
793 We wish to acknowledge the generous support of Dr.Sanjay Sane in graciously allowing us the
794 use of his flight column, Dr.Gaiti Hasan for the itpr alleles and the learning of specific protocols
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795 and NCBS, Bangalore in general, for facilitating the availability of various fly stocks. Assistance
796 provided by Andrew Orry of Molsoft, (San Diego, CA, USA) in our homology modeling efforts
797 is appreciated. We gratefully acknowledge Dr.R.Baskar and Dr.Madhulika Dixit of the
798 Department of Biotechnology at IIT Madras respectively for microscopy facilities and assistance
799 provided for some of the RT-PCR experiments [including specific funding made available
800 through a grant from the Department of Science and Technology (SR/FT/LS-123/2008)]. This
801 research was partially funded in its very initial stages by a grant from the Department of
802 Biotechnology to Dr.A.Gopalakrishna (BT/IN/FRG/10/AGK/2007) and in its later stages, to a
803 limited extent, by grants from the IIT Madras Research Foundation.
804
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959
960
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961 List of Figure Captions
962
963 Figure 1: The dmnucb1c01508 mutant and dmnucb1 RNAi lines are valuable tools to uncover
964 functional roles for Drosophila NUCB1, whose modularity is largely conserved.
965 Figure 2: A single copy of the dmnucb1c01508 allele rescues wing posture defects in itpr mutants
966 and augments restoration of flight by dOrai.
967 Figure 3: The heterozygous dmnucb1c01508 mutant alleviates cold-sensitive lethality in itpr
968 mutants.
969 Figure 4: dmnucb1 can modulate the Gαq-Phospholipase Cβ-Ca2+ pathway in the gut.
970 Figure 5: Rescue of TDP-43 associated motor neuron phenotypes by itpr mutants is reversed by
971 dmnucb1 disruption in tandem. Draft
972 Figure 6: Important amino acid determinants in vertebrate NUCB1 that enable its docking onto
973 the hydrophobic binding cleft of Gαi3 are also conserved in Drosophila, but completely
974 disrupted in the dmnucb1c01508 mutant.
975
976
977
978
979
980
981
982
983
984
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985 Figure Captions and Legends
986
987 Figure 1: The dmnucb1c01508 mutant and dmnucb1 RNAi lines are valuable tools to
988 uncover functional roles for Drosophila NUCB1, whose modularity is largely conserved.
989 A. Structure of the dmnucb1 locus, encoding a single transcript of roughly 2.2kb. The
990 homozygous-lethal dmnucb1c05934 allele is shown merely for comparative reference. B.
991 Schematic showing the predicted protein structure for Drosophila NUCB1 and its various
992 constituent domains, motifs or signals. [SP: Signal Peptide; GRS: Golgi Retention Sequence;
993 EF1-3: EF hand motifs 1, 2 and 3, respectively; AR: Acidic Region. The ‘Proline+2’ ER export
994 signal – 2 amino acids past the signal peptide – is indicated by a thin, dark line with a ‘+2’
995 notation on top]. C. Chromatogram showingDraft the exact insertion point (downward pink arrow) of
996 the pBac[PB]NUCB1[c01508] element, immediately prior to the first predicted residue of the 7
997 amino acid GBA core. D. Semi-quantitative PCR analyses confirm a significant reduction in
998 dmnucb1 expression in both RNAi lines. Approximately 34% and 44% (reduced) transcription
999 levels are seen for the tubulin-Gal4 > UAS-dmnucb1RNAi(X) and tubulin-Gal4 > UAS-
1000 dmnucb1RNAi(3) stocks, respectively, relative to wild-type expression levels. dmnucb1
1001 expression is however not significantly compromised in the dmnucbc01508 mutant allele. Results
1002 from 3 independent trials are shown. g3pdh / GAPDH control expression levels are shown
1003 alongside each genotype, to ascertain dmnucb1-specific knockdown. Error bars indicate SEM.
1004 Orientation of primers used for genomic flanking sequence determination and various RT-PCR
1005 experiments are indicated with dented arrows in Fig. 1A above and detailed further, with DNA
1006 sequences, in Supplementary Table ST528. A dmnucb1-specific (exon 2) reverse primer was
1007 used in conjunction with a forward primer corresponding to the piggyBac parent vector to
28 Supplementary Table ST5
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1008 reconfirm the genomic sequence flanking thec01508 insertion, in addition to the inverse PCR-
1009 based approach. It is alone hence shown with a dented thicker arrow (in pink and marked with
1010 an asterisk).
1011
1012 Figure 2: A single copy of the dmnucb1c01508 allele rescues wing posture defects in itpr
1013 mutants and augments restoration of flight by dOrai.
1014 A. A single copy of the c01508 transposon insertion completely rescues the ‘spread out’ wing
1015 posture defect in both itpr heteroalleles analyzed. Excision of the transposon causes reversion of
1016 wing posture to the mutant phenotype (dmnucb1c01508Rev1R-itprug3 / itprwc703). B. The homozygous
1017 dmnucb1c01508 allele is seen to improve flight behavior by roughly 19% and 21% respectively, in
1018 the dOrai11042 / +; itprug3 / itprwc703 andDraft dOrai11042 / +; itprug3 / itprwc361 double mutants. Almost
1019 76% of the Kum170 / +; dOrai11042 / +; itprug3-dmnucb1c01508 / itprwc703-dmnucb1 c01508 and 77%
1020 Kum170 / +; dOrai11042 / +; itprug3-dmnucb1c01508 / itprw361-dmnucb c01508 quadruple mutant flies
1021 respectively, show normal flight behavior. The homozygous dmnucb1c01508 allele is also seen to
1022 improve flight behavior by roughly 16% and 15%, and the heterozygous allele by roughly 10%
1023 and 9%, respectively in the Kum170 / dOrai11042; itprug3 / itprwc703 and Kum170 /
1024 dOrai11042; itprug3 / itprwc361 triple mutants. Excision of the piggyBac insertion nullifies this
1025 improvement (stocks labelled dmnucb1c01508Rev1R and dmnucb1c01508Rev2R). C. Induction of either
1026 dmnucb1 RNAi line in aminergic neurons phenocopies the improvements in flight behavior, of
1027 dOrai11042 / +; itprug3 / itprwc703 and dOrai11042 / +; itprug3 / itprwc361 double mutants by
1028 dmnucb1c01508 (16% in both cases). Neither RNAi line [dmnucb1 RNAi(X) and dmnucb1
1029 RNAi(3)] by itself displayed noticeable flight impairment when driven by Ddc-Gal4. Statistical
1030 significance is indicated using t tests (*, #, π, ♦), where it may not be visually obvious. Error
1031 bars indicate SEM.
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1032
1033 Figure 3: The heterozygous dmnucb1c01508 mutant alleviates cold-sensitive lethality in itpr
1034 mutants.
1035 At roughly 576 hours after egg laying, only 6% of itprug3 / wc703 and 29% of itprug3 / wc361 mutants
1036 respectively, survive at 17.5°C, whereas the introduction of a single copy of dmnucb1c01508
1037 dramatically increases their survival percentages to 82% and 86%, respectively (A, B). These
1038 percentages were only marginally increased in the case of dmnucb1c01508 homozygotes (84% and
1039 88%, respectively). Stocks in which the c01508 insertion was excised – dmnucb1c01508Rev1R and
1040 dmnucb1c01508Rev2R – displayed an equally dramatic reversion to the cold-sensitive phenotype.
1041 The parent dmnucb1c01508 stock was fully viable at 17.5°C, even in the homozygous state. Error
1042 bars indicate SEM. Draft
1043
1044 Figure 4: dmnucb1 can modulate the Gαq-Phospholipase Cβ-Ca2+ pathway in the gut.
1045 Log rank analyses reveal that while 50% of NP3084-Gal4 > UAS-PLCβ-RNAi flies remain alive
1046 on ‘live yeast’ media by day 30, a single copy of the dmnucb1c01508 mutant increases the same
1047 survival fraction by five days (A). Viability profiles were only slightly further enhanced in the
1048 case of the homozygous dmnucb1c01508 / c01508 allele (B). Gut-specific knockdown of dmnucb1 in
1049 tandem with UAS-PLCβ-RNAi also shows an augmentation of survival on ‘live yeast’ media,
1050 with either RNAi line, albeit to a lesser degree [50% survival at 34 days in the case of UAS-
1051 dmnucb1 RNAi(X) and at 33 days for dmnucb1 RNAi(3): C, D]. Excision lines lacking the
1052 transposon insertion failed to rescue the gut immunity defects seen in NP3084-Gal4 > UAS-
1053 PLCβ-RNAi flies. Both RNAi lines showed near wild type viability profiles, when driven by
1054 NP3084-Gal4, on yeast supplemented media. P-value < 0.0001, as determined by log rank
1055 analyses.
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Page 48 of 49
1056
1057 Figure 5: Rescue of TDP-43 associated motor neuron phenotypes by itpr mutants is
1058 reversed by dmnucb1 disruption in tandem.
1059 A. A single copy of the dmnucb1c01508 insertion decreases by 22%, the climbing index of flies
1060 overexpressing human TDP-43 against the (otherwise dominant rescue) background of itprug3.
1061 Such regression is also phenocopied to the tune of 18% and 14% respectively, by independently
1062 co-expressing two different dmnucb1 RNAi lines in motor neurons. Dominant rescue of climbing
1063 defects in D42-Gal4 > UAS-TDP-43 flies by the itpr90B.0 null allele is overturned by roughly
1064 13%, when either dmnucb1 RNAi is co-induced in motor neurons. t tests were performed for
1065 comparing the results from each genotype. Error bars indicate SEM. B. The heterozygous
1066 dmnucb1c01508 allele regresses by fiveDraft days, the lifespan improvement achieved by itprug3 in
1067 D42-Gal4 > UAS-TDP-43 flies, at the 50% survival mark. This effect is phenocopied in almost
1068 equal measure by flies co-expressing either RNAi line in motor neurons. Reversion of both
1069 modifier effects wrought by the dmnub1c01508 allele were evidenced in dmnucb1Rev1R and
1070 dmnucb1Rev2R (A and B). On account of highly limiting chromosomal constraints underlying
1071 these genetic crosses, the homozygous insertion could not be assayed in both experiments.
1072 Neither RNAi line by itself showed locomotor or lifespan defects, when driven by D42-Gal4. P-
1073 value less than 0.05, as determined by log rank analyses.
1074
1075 Figure 6: Important amino acid determinants in vertebrate NUCB1 that enable its docking
1076 onto the hydrophobic binding cleft of Gαi3 are also conserved in Drosophila, but
1077 completely disrupted in the dmnucb1c01508 mutant.
1078 A. Homology modeling of the GBA motif in dmNUCB1 (in red) using the human NUCB1 GBA
1079 sequence [in blue, coordinates extracted from the Protein Data Bank (PDB#: 1SNL)] and the
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1080 KB-752 synthetic peptide (in green; PDB#: 1Y3A) complexed with human Gαi1 (cyan
1081 backbone) as templates, reveals that the putative interaction of NUCB1 with Gαi subunits is
1082 likely conserved to a high degree, in Drosophila. Boxed inset compares sequences from the
1083 GBA core of dmNUCB1 with orthologous sequences from KB-752 and human NUCB1, against
1084 the consensus 7aa sequence for the GBA motif. Ψ represents aliphatic and Φ aromatic, residues.
1085 Interaction-critical residues 3, 6 and 7 from the GBA consensus sequence are also indicated, as
1086 Φ(3), F(6) and Ψ(7) (in black). B. Interaction-critical residues from the GBA motif of
1087 dmNUCB1 are shown schematically juxtaposed with partnering amino acids from the α3 /
1088 Switch II region (labeled in pink) of human Gαi. Residues in positions 3, 6 and 7 of the
1089 dmNUCB1 GBA core sequence (I309, F312 and M313; indicated in red) correspond to
1090 hydrophobic residues aligned on one sideDraft of the α-helical part of the motif. These residues are
1091 proposed to stabilize the interaction with Gαi, by packing against the W211 and F215 residues
1092 (indicated in pink) of the α3 / Switch II hydrophobic cleft (ribbons in cyan). C. Modeling
1093 analyses further predict the K248 residue (in pink) of Gαi to be located in close proximity to
1094 D310 (in red) of dmNUCB1, suggesting – as in vertebrates – that they may together specify an
1095 important interaction point, based on electrostatic attraction [also refer Appendix Fig. A4(a)]. D.
1096 The c01508 transposon insertion (downward red arrow) completely disrupts the GBA motif core
1097 that follows (7aa underlined & highlighted in blue), by introducing 3 in-frame stop codons
1098 [underlined ‘TAA’ in red, with yellow highlights (top panel) and translated as red asterisks in
1099 the cognate protein sequence (bottom panel)]. In addition to these stop codons, complete lack of
1100 conservation is observed over a 20aa stretch beyond the c01508 insertion, including the GBA
1101 core.
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Draft
Figure 1: The dmnucb1c01508 mutant and dmnucb1 RNAi lines are valuable tools to uncover functional roles for Drosophila NUCB1, whose modularity is largely conserved.
95x125mm (300 x 300 DPI)
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Draft
Figure 2: A single copy of the dmnucb1c01508 allele rescues wing posture defects in itpr mutants and augments restoration of flight by dOrai.
88x72mm (300 x 300 DPI)
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Draft
Figure 3: The heterozygous dmnucb1c01508 mutant alleviates cold-sensitive lethality in itpr mutants.
52x85mm (300 x 300 DPI)
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Draft
Figure 4: dmnucb1 can modulate the Gαq-Phospholipase Cβ-Ca2+ pathway in the gut.
85x85mm (300 x 300 DPI)
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Draft
Figure 5: Rescue of TDP-43 associated motor neuron phenotypes by itpr mutants is reversed by dmnucb1 disruption in tandem.
87x89mm (300 x 300 DPI)
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Draft
Figure 6: Important amino acid determinants in vertebrate NUCB1 that enable its docking onto the hydrophobic binding cleft of Gαi3 are also conserved in Drosophila, but completely disrupted in the dmnucb1c01508 mutant.
129x140mm (300 x 300 DPI)
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Draft APPENDIX
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Background Information
Nucleobindin-1 (also known as NUCB1 or Calnuc) is a multi-domain EF-hand calcium-
binding protein, phylogenetically conserved from worms to humans (Aradhyam et al. 2010).
It has been identified as the major Golgi-associated calcium (Ca2+) binding protein and to be
involved in the establishment and maintenance of an agonist-mobilizable Golgi Ca2+ store, in
a wide variety of vertebrate cell and tissue types (Lin et al. 1998; Lin et al. 1999).
Vertebrate NUCB1 has been shown to harbor a motif of 15-25 amino acids (aa) that can
directly bind to G protein α subunits and stimulate their guanine nucleotide exchange activity,
without the involvement of a G protein coupled Receptor (GPCR), and at the cytoplasmic
surface of Golgi membranes (Aznar et al. 2016; Garcia-Marcos et al. 2011). Among the small
class of proteins currently known to containDraft this G protein α-subunit Binding and Activating
(GBA) motif, vertebrate NUCB1 and its paralog NUCB2 are the only members known to
bind Ca2+ and the only ones localizing primarily to the Golgi. Indeed, since the GBA motif in
these proteins overlaps with one of their EF hands, binding of Ca2+ has been shown to abolish
their respective interactions with Gαi1/3 subunits (Garcia-Marcos et al. 2011).
Objectives
This stand-alone document intends to catalogue, mostly at the primary amino acid sequence
level, the extent of conservation of multiple important features characterizing:
a) Potential NUCB1-Gα subunit interactions in select invertebrate organisms, based on the
sequence / residue-specific biochemical validation of rat NUCB1 / Calnuc binding [and / or
independently, rat NUCB2 / NEFA] to Gαi1 or independently, Gαi3 [(Garcia-Marcos et al.
2011) Figs. A1, A3, A4(a), A4(b) and Table AT2], and,
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b) The potential transport to, or localization to the Golgi, of NUCB1, in these select
invertebrate organisms, based on information derived from cells in culture, involving human
NUCB1 or NUCB2 (Figs. A5-7).
It also aims at documenting the potential lack of conservation (by contrast) among
invertebrates, of a C-terminal region that was originally named the ‘PVQ5 repeat’ region,
more than 20 years back [in the pre-genome sequencing era, based on predicted sequence
information derived from Drosophila melanogaster NUCB1 (henceforth dmNUCB1) alone
(Otte et al. 1999)].
Organisms analyzed
Other than Drosophila melanogaster (henceforth, D.melanogaster) – the experimental
organism used in our studies – 5 otherDraft invertebrate organisms were selected for comparative
sequence analyses, as follows:
1) From among the 12 Drosophila species originally chosen for whole genome sequencing
[publicly available information for which has been progressively annotated on FlyBase
(www.flybase.org) over the last decade and more, (Clark et al. 2007)]:
Drosophila simulans (henceforth, D.simulans), as the closest phylogenetic neighbor to
D.melanogaster,
Drosophila virilis (henceforth, D.virilis), as being far more distant to D.melanogaster
on the Drosophila phylogenetic tree than D.simulans, but sharing a worldwide
distribution pattern with D.simulans, and,
The cactophilic, desert-inhabiting Drosophila mojavensis (henceforth, D.mojavensis)
on the one hand and the Hawaiian volcanic island resident Drosophila grimshawi
(henceforth, D.grimshawi), on the other. These last two organisms were selected as
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examples of species that are also relatively more distant** to D.melanogaster on the
phylogenetic tree, but are additionally distinguished by restricted habitats.
**D.grimshawi is in fact, the farthest Drosophila species from D.melanogaster,
among those species for which whole genome sequence information is publicly
accessible.
2) The nematode (or worm) Caenorhabditis elegans (henceforth, C.elegans) as an
alternatively popular invertebrate model organism for genetic experimentation.
Methods used
The tBLASTx search algorithm with default parameters and the non-redundant
protein sequences reference database were used in all multiple sequence alignment
analyses, as made Draft available through the NCBI portal
(https://blast.ncbi.nlm.nih.gov/Blast.cgi). Updated sequence information for
individual predicted proteins from D.mojavensis & D.grimshawi is no longer provided
by FlyBase. Gene model annotations for these species and were extracted instead,
through the NCBI gnomon automated annotation pipeline
(https://www.ncbi.nlm.nih.gov/genome/annotation_euk/all/).
Interaction parameters shown in Table AT2 were derived using the ICM Browser Pro
software (Molsoft, San Diego, CA, U.S.A., www.molsoft.com)
Data representation
a) Fully conserved residues are indicated in red (and a ‘*’ at the bottom of each sequence set),
highly conservative substitutions in green (and a ‘:’ at the bottom of each sequence set) and
less conservative substitutions in light blue (and a ‘.’ At the bottom of each sequence set, in
all figures that follow.
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b) Multiple sequence alignments are shown for all isoforms (NUCB1, Gαi, Gαoa-b) encoded in each of 6 invertebrate species, against human NUCB1 and Gαi1, respectively. Human
NUCB2 / NEFA, Rat NUCB1 and rat NUCB2 / NEFA have been included in each alignment set for the sake of completeness of analysis. It must be noted however, that when alignments are done purely between the 5 chosen Drosophila sequences, significantly higher degrees of conservation are observed for the GBA motif [Fig. A4(b)], the 3 EF hands (Fig. A3), the
Golgi Retention Sequence (Fig. A6) and the membrane lipid anchoring motif (Fig. A7).
Overall Conclusions
Amino acids from the G protein α-subunit Binding and Activating (GBA) motif 7aa
core, experimentally validated to critically specify the binding of NUCB1 with Gαi1/3
subunits in vertebrates, are highlyDraft conserved in D.melanogaster, at the levels of
primary sequence, as well as structural conformation.
Sequence-level conservation also holds true for 4 other Drosophila species (regardless
of their evolutionary distance from D.melanogaster and whether or not they occupy
restricted habitats or have come to colonise the world), and independently, the
nematode C.elegans, too.
In addition, motifs or signals that have been experimentally shown in vertebrates to
determine the transport to (Proline+2), or association with, the Golgi (Golgi Retention
Sequence or GRS), are also seen to be appreciably conserved across all 6 invertebrate
species. An N-terminal motif that has been shown to anchor vertebrates Gαi1/3
subunits to membrane lipids is similarly conserved to a high degree in all 6
invertebrate species examined.
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In sharp contrast, the C-terminal region of NUCB1 varies considerably across all 6
invertebrate NUCB1 predicted sequences and does not easily lend itself to uniform or
definitive characterization.
References
Complete journal information and other associated details for all references cited, are listed at
the very end of this document.
Note
Wherever it has not been possible to accommodate legends with the corresponding figures or
tables on a single page, the terms ‘Figure overleaf’ or ‘Table overleaf’ have been added, to
connote that the figure or table meantDraft to accompany any given legend, may be found on the
following page.
******************************************
FIGURES & TABLES WITH CORRESPONDING LEGENDS
Figure A1: The motif / domain-rich modularity of NUCB1 is mostly conserved across
evolution from worms to humans, including 5 different Drosophila species.
SP: Signal peptide (boxed in purple); +2 P: Proline+2 ER export signal (indicated by a thin,
dark line); GRS: Golgi Retention Sequence (boxed in yellow); AR: Acidic amino acid
Region (boxed in gray); EF1-3: EF hands 1, 2 & 3 respectively (all boxed in green); LZ:
Leucine Zipper (boxed in pink). The basic amino acid region (boxed in turquoise) and the
GBA motif (boxed in light blue, overlap with EF2 indicated by a blue bridge / bent arrow) are
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also shown. Amino acid correlates for all signals, motifs or domains are shown diagonally alongside to indicate that most of the modularity seen in vertebrate NUCB1 or NUCB2 is conserved at roughly equivalent positions. 5 of the 6 invertebrate NUCB1 predicted sequences represent single isoforms, while D.simulans alone encodes 2 isoforms (differing in merely 3 amino acids, throughout the length of the corresponding predicted protein sequences; the larger isoform is alone shown here). All 6 invertebrate sequences lack the leucine zipper domain and contain instead, a ‘Variable repeat region’ (boxed in brown at the
C-terminus, refer to Fig. A2(a) and Table A1 below, for more details). The overall length of invertebrate NUCB1 predicted protein sequences vary considerably in length in comparison with each other or human NUCB1. While the predicted invertebrate proteins are much longer than human NUCB1 in some cases, (108aa longer in D.melanogaster; 103aa and 106aa longer in D.simulans and 116aa longerDraft in D.virilis), they are relatively more comparable in length to human NUCB1, in other cases (only 38aa longer in D.mojavensis, 52aa longer in
D.grimshawi and only 6aa longer in C.elegans).
[Figure overleaf]
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Figure A1
Draft
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Figure A2: The ‘PVQ5 repeat’ nomenclature proposed earlier for D.melanogaster
NUCB1 inaccurately reflects the content of this region when assessed across 6 different
invertebrate species.
The number of predicted PVQ repeats in the C-terminal region of NUCB1 vary widely across
the 6 invertebrate sequences analyzed. Raw predicted sequence information is shown for all 6
organisms, (as opposed to multiple sequence analysis, given the pervasive variability inherent
across this region). The ‘variable repeat region’ was marked to start in D.melanogaster with
the first stretch of 3 or more ‘Q’ residues and this starting point was cross-referenced in other
species by manual curation. ‘Q’ repeats are highlighted in yellow and ‘PVQ1-5’ repeats are
underlined. The occurrence of variant repeats such as PVYQ are also shown with dotted
underlining, in some species, wherever they may be found The only other trinucleotide repeat
(PPP) in the entire C-terminal regionDraft is shown in red highlights. The two isoforms of
D.simulans are identical to each other with respect to their C-terminal regions. Comparisons
of the Watterson’s θ, Tajima’s π and Tajima’s D genetic variation parameters based on the
PoPoolation database (Pandey et al. 2011), signature patterns from the RepeatsDB database
(Hirsh et al. 2018) or genomic polymorphisms from this region using the FlyVar database
(Wang et al. 2015) failed to yield meaningful insights. It is also unclear whether replication
slippage may have played a role in shaping the frequency of occurrence of ‘Q’ repeats in the
C-terminal region of NUCB1 from various invertebrates or whether purifying selection may
have come into play for these repeats or their flanking regions, during evolution. Analyses
leveraging Drosophila Genetic Reference Panel (DGRP) lines may conceivably be more useful in identifying specific haplotype groups corresponding to this hypervariable repeat region.
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Taken together with the data collated in Table AT1 (below), the ‘PVQ5’ nomenclature
proposed earlier for this region (Otte et al. 1999) is largely inaccurate. We have therefore re-
named this region more accurately as the ‘Variable repeat region’ (VRR).
D.melanogaster NUCB1
402QQQQQYAQQQQQYAQQYQQQQYGNGQQPVQLQPNQVYQHAGQIPQQQQPVYQNQPVYQQQ QPVYQQQQPVQQQQKPVQQPVQQQQQPVQQQQQPVQQQQQTVQQQQPVQQQQQTVQQQQP VQQQQQTAQQQPVAQQQIHNQSPPPVLNQQVPVQQQQKQHQESLNQQH569
D.simulans NUCB1
405QQQQQYAQQQQQYAQQYQQQQYGNGQEPVQLQPNQVYQHAGQIPQQQQPVYQNQPVYQQQ QPVYQQQQPVQQQQQPVQQQQQPVQQHQQPVQQQQQPVQQQQQPVQQQQQTVQQQQQTAQ QQPVAQQVHNQSPPPVQNQQVPVQQQQKQHQESLNQQH562 D.mojavensis NUCB1 Draft 398QQQQQYHHQQQQYAQQQQQQYGGSQPVPAYQHQQPQPLIYQQPSQQQQPLPTYRHVQHNG HPEQVSLLQQSNSETPSLVQKQQPQDKLQNVHTTSAKIPTTK499
D.virilis NUCB1
402QQQQQYEHQQQQYAQQQQNHGQQYQQQQYGQPQPVQLNPDQVYQHAGQIPEHQQAYQQQQ QPLIYQQPSQQQPSQQQPVYQPVQPAVQPGQVPAQQQQQPVYQPVQPAVQPGQVPAPQQQ QPAQQQAHQQINNQSPSPQQNQQLPVQQQQQQSKDQVLQHDPSKQQQKPEQQKLQH577
D.grimshawi NUCB1
399QQQQQYAHQQQQYAQQQQQYNEPYQHQQNAQPQPVQLNPNQVYEHAKQTPEGLQVNQQPI QMQQKQQQQPVQQQPLQQQQHTEGQNQVNLQQANNQSTPFVQNKQPQQIPEAQHH513
C.elegans NUCB1
397QPPQAQQQVHPAQQPIQPVNANPPPVQNAQPPVQQQQQPPQQPPQQPPQQNLPPVHHEPI QDHTKDPTYGI467
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Table AT1
The Variable Repeat Region differs considerably across invertebrate NUCB1 homologs,
with respect to multiple criteria examined.
Striking differences for each of 5 other invertebrate predicted NUCB1 sequences, in
comparison with the repeat region of the reference dmNUCB1 sequence, are indicated in
bolded pink and relatively less dramatic differences, in bolded green. Pertinent examples
include:
a) The VRR of C.elegans and D.mojavensis are 97aa and 60aa shorter, respectively, than that
in dmNUCB1,
b) D.mojavensis harbors zero PVQ repeats while D.grimshawi and C.elegans harbor only 2 repeats each, in comparison to 9 repeats each, in dmNUCB1 and NUCB1 from D.simulans.
Percentages of constituent amino acidsDraft from this region also vary considerably across the invertebrate sequences examined, as indicated and in contrast to, for example, the Glutamine- rich hypervariable N-terminal repeat regions in the Argonaute-2 domain containing protein family (Palmer and Obbard 2016). Almost double and more than 1.5 times the percentage of glutamine residues are found in C.elegans and D.mojavensis, respectively, when compared with the VRR of dmNUCB1. C.elegans has more than 2.5 times the percentage of prolines as dmNUCB1 and both D.mojavensis and D.grimshawi contain less than half the percentage of valines found in the VRR of dmNUCB1. Many other constituent amino acids also show dramatic comparative shifts, beyond just the ‘P’, ‘V’ or ‘Q’ residues.
[Table overleaf]
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Table AT1
D.melanogaster D.simulans D.mojavensis D.virilis D.grimshawi C.elegans
Variable Region 168 aa 158 aa 102 aa 176 aa 115 aa 71 aa Length
Number of PVQ (1-5) 9 9 0 4 2 2 Repeats
Number of PVYQ 3 3 0 2 0 0 Repeats
Number of Poly Q 17 14 4 12 6 2 Repeats (≥3)
% Q 55.36 55.06 34.31 46.02 42.61 30.99
% P 10.71 10.76 10.78 13.64 9.57 28.17
% V 10.71 10.76 4.90 6.82 5.22 7.04
% Y 4.76 5.06Draft5.88 5.11 4.35 1.41
% N 3.57 3.80 2.94 2.84 8.70 5.63
% A 2.98 3.16 2.94 5.11 5.22 5.63
% H 2.38 3.16 6.86 3.98 5.22 5.63
% L 1.79 1.27 5.88 2.84 3.48 1.41
% T 1.79 1.27 5.88 0.0 2.61 3.03
% I 1.19 0.63 1.96 1.70 1.74 4.22
% S 1.19 1.27 6.86 3.41 0.87 0.0
% K 1.19 0.63 3.92 2.27 2.61 1.41
% E 0.60 1.27 1.96 1.70 4.35 1.41
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Figure A3: EF-hand 1 (EF1) and the proposed EF-hand 3 (EF3) are more stringently conserved in NUCB1 orthologs, than EF-hand 2 (EF2).
A. When the single dmNUCB1 isoform is compared with its rat and human counterpart(s),
17 and 15 residues out of 29, respectively (58% and 51%), are identically conserved in
EF1 (a) and EF3 (c). By contrast, only 11 amino acids (37%) are identically preserved in
EF2 (b). This trend is maintained across all 4 other Drosophila species analyzed, but not
in C.elegans. EF1 and EF3 may thus have been under relatively more stringent
evolutionary selection pressure than EF2, after the divergence of insects from worms. The
noncanonical EF3 hand motif has only been proposed for Drosophila NUCB1 and
homologous regions from other species are shown for comparison, although EF3 has not
been experimentally validated for calcium binding in any organism shown. The seven 7aa
core sequence of the GBA motif isDraft also indicated as overlapping with EF2 (labeled box
within the loop region). B. Schematic depicting the organization of EF2. O: Oxygen
atom–containing side chains, Ψ: Hydrophobic amino acid, G: Canonical Glycine residue.
The transposon insertion in a stock publicly available through FlyBase (www.flybase.org,
PBac[PB]NUCB1[c01508] or dmnucb1c01508) is shown as completely disrupting the loop
region of EF2 and also the contiguous GBA motif. This stock has been preferentially used
in most of our experiments and results thereof are documented in detail in the main
manuscript text.
It has been observed that in rat and human NUCB1, EF- hand 1 (EF1) is the main Ca2+- binding site and that EF2 has a significantly lower affinity for Ca2+ ions than EF1 (Kapoor et al. 2010; Lin et al. 1999). EF3 – or conceivably, the (S100-like) pseudo EF hand sequence detected upstream of EF1 [not shown here, (Zhou et al. 2006)] – may functionally substitute for EF2 in the dmnucb1c01508 mutant allele, although this needs to be experimentally validated
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in either case. EF2 may however minimally bind Ca2+ ions to negatively regulate GBA
function, as required in the wild type. It must however also be noted that the difference in
binding affinities between EF1 and EF2 in human Calnuc could conceivably arise from the
presence of a noncanonical Arginine residue substituting for the canonical Glycine at the
sixth position of the 12-residue Ca2+ binding loop. A canonical Glycine is however found at
this position in dmNUCB1.
[Figure overleaf]
Draft
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Draft
Figure A4(a): Near-complete conservation of residues crucial for functional linkage with NUCB1, in invertebrate Gαi or Gαo subunits.
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The 13 amino acid Switch II region (shown mostly in red) is identically conserved in
its entirety, across evolution, from the C.elegans GOA-1 or Gαi/o subunit [Q to E]
(Cuppen et al. 2003) to single Gαi isoforms in each of 5 different Drosophila species
analyzed [uniformly Q205 to E217] and 3 different isoforms in the rat and human
counterparts [Q204 to E216 of Gαi1-3, in both organisms].
W211 and F215 in the Switch II / α3 cleft from rat Gαi1/3 have been postulated on
the basis of biochemical analyses, to mediate hydrophobic interactions with the L313,
F316 and L317 residues of the GBA core in rat NUCB1 (Garcia-Marcos et al. 2011).
Identical invertebrate equivalents of these amino acids are shown in numbered yellow
[W212 and F216 in all 5 Drosophila species, alike and C.elegans].
K248 in the middle of the α3 helix (also shown mostly in yellow) has been identified
in rat Gαi1/3 to electrostaticallyDraft interact with E314 of rat NUCB1. Mutation of this
lysine to methionine in rat Gαi3 has been shown to dramatically decrease its ability to
bind rat NUCB1 and mutation of M249 to K249 in rat Gαo, to conversely enhance
binding to rat NUCB1 (Garcia-Marcos et al. 2011). The preferential binding of Gαi
over Gαo by rat NUCB1, is thus partly determined by this positively charged lysine.
(Garcia-Marcos et al. 2011). This residue is indicated in numbered yellow, as being
conserved in all 5 Drosophila species and C.elegans (uniformly K249), as well. W258
in the α3/β5 loop has also been similarly shown to dictate the preferential binding of
Gαi over Gαo by another vertebrate GBA motif-containing protein, GIV / Girdin, but
to not be important for rat Gαi1/3 to rat NUCB1. This residue (again, in numbered
yellow) is nevertheless also identically retained in all invertebrate sequences
examined [W259 in all 5 Drosophila species and in C.elegans].
Interestingly however, K249 is also conserved in both isoforms of Gαo (Gαoa/b) in
D.melanogaster, D.simulans, D.mojavensis and D.virilis, in the single Gαo isoform
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from D.grimshawi and in C.elegans GOA-1, as well. Gαo isoforms from all of these
organisms are identical to each other across the entire Switch II-α3 region, including
the W212, F216 and W258 residues. In invertebrates therefore, interaction of NUCB1
with G protein α subunits may be relatively more promiscuous and the Gαi over Gαo
binding preference may have been acquired later in evolution.
[Figure overleaf]
Draft
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Figure A4(a)
Draft
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Figure A4(b): Significant conservation of multiple binding determinants in the GBA motif core of invertebrate NUCB1.
Sequences corresponding to the extended GBA motif in human NUCB1 and NUCB2, rat
NUCB1 and NUCB2, five different Drosophila species and C.elegans are shown.
Invertebrate equivalents of critical amino acids L313, F316 and L317 {positions 3, 6
and 7 of the seven amino acid (aa) core GBA sequence ( [Ψ]-[S/T]-[Φ/Ψ]-x-[D/E]-
[F]-[Ψ] where Ψ represents aliphatic and Φ aromatic, residues) required for NUCB1-
Gαi1/3 interaction in vertebrates (Garcia-Marcos et al. 2011), are shown in
highlighted yellow and with numbered arrows. These residues are conservatively
represented by I309, F312 and M313 in D.melanogaster (also refer to Fig. 6B of main
manuscript text) and I, F and M residues in equivalent positions in 3 of the 4 other
Drosophila species analyzedDraft (D.simulans, D.virilis and D.grimshawi). In
D.mojavensis, I309 is represented instead by M308 and in C.elegans, these 3 amino
acids are represented by M327, F330 and L331, respectively.
T312 and E315 (positions 2 and 5 in the GBA core) are postulated in rat NUCB1 to
form a hydrogen bond that allows the GBA motif to assume a helical conformation
(Garcia-Marcos et al. 2011). These are likewise conserved as S308 and E311
respectively, in D.melanogaster, as also in all 4 other Drosophila species and worm
NUCB1, alike (depicted with underlining squiggles).
The negatively charged E314 from rat NUCB1, which has been postulated to
electrostatically interact with K248 of rat Gαi3 (Garcia-Marcos et al. 2011) is
conserved as D310 in D.melanogaster [negatively charged aspartic acid (D), instead
of glutamic acid (E); also refer to Fig. 6C of main manuscript text] and as,
respectively, D310 and D312 in the two isoforms of D.simulans, D309 in
D.mojavensis, D310 in D.virilis and E307 in D.grimshawi (all underlined). In
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C.elegans NUCB1 however, this residue is replaced by a neutrally charged glutamine
(Q328, shown with an underlining ‘…’ notation). The interaction of worm Gαi/o or
GOA-1 with worm NUCB1 has nevertheless been validated in a yeast two-hybrid
assay (Cuppen et al. 2003). RNAi analyses however failed to produce any discernible
phenotypes, in these experiments (Cuppen et al. 2003).
Taken collectively [Figs. A4(a) and A4(b), above], it appears reasonable to hypothesize that
the interaction between Gαi1/3 and NUCB1 experimentally validated in vertebrates, is likely
conserved at a structural and subcellular level, in multiple invertebrate organisms. 10
different determinants from the primary sequence level, as also related homology modeling
parameters (jointly encompassing both sides of the interaction; Table AT2, below) thus lay
the groundwork for biochemically furtherDraft confirming the NUCB1-Gαi subunit interaction in
D.melanogaster, with the significant caveat that dmNUCB1 may additionally bind to
dmGαoa/b.
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Table AT2: Conformational modeling of dmNUCB1 confirms the structural
conservation of residues critical for its interaction with Gα subunits.
Quantitative estimates of various interaction parameters are shown, as derived from
homology modeling of the 7aa core sequence of the dmNUCB1 GBA motif against the 3D
coordinates for human NUCB1 [obtained from the Protein Data Bank (PDB#: 1SNL)], in turn
threaded over the structural coordinates of the synthetic peptide KB-752 complexed with
human Gαi1 (PDB#: 1Y3A) (de Alba and Tjandra 2004; Johnston et al. 2005). ‘Contact
Area’ refers to the area of any given residue in contact with its interaction partner (Å2);
‘Exposed Area’ refers to area of any given residue exposed to solvent (Å2); ‘Closest
Distance’ refers to distance in angstroms from any given residue to the interaction partner.
Modeling analyses predict that putativeDraft interactions between dmNUCB1 and Gα subunits are
likely to be driven primarily by hydrophobic interactions between F312, M313, I307 and
L306, from the GBA core motif and the fly amino acid equivalents of F215 and W211 of the
human Gαi1 subunit. This table however highlights a significantly larger subset of amino
acids analysed collectively in dmNUCB1 and human Gαi1, in comparison with the most
important ones depicted in Figs. 6A-C of the main manuscript text.
[Table overleaf]
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Table AT2
dmNUCB1(aa) Contact Exposed Area Percent Closest Area Buried Distance
I309 91.977448 133.756943 69 1.550709 F312 82.72184 121.828705 68 2.057679 M313 69.634315 131.137695 53 2.336698 I307 66.377808 109.427719 61 1.800313 L306 47.475883 140.387711 34 2.45172 G305 22.413319 66.964691 33 2.907497 D310 20.051823 78.440369 26 3.536902 S308 19.105103 62.609009 31 2.664929 Q315 15.923576 110.08326 14 2.344904 D304 14.27066 153.880539 9 2.421042 V314 6.904427 106.231148 6 3.845757
Human Gαi1 (aa) Contact DraftExposed Area Percent Closest Area Buried Distance
R208 76.846344 168.412872 46 2.067085 W211 62.564621 94.156441 66 1.721612 R205 55.875427 202.782684 28 2.067161 F215 46.042625 100.77079 46 2.589464 S252 32.766071 76.396194 43 2.97453 S206 31.402176 66.531281 47 1.797546 E207 18.195778 106.047249 17 2.63133 I253 17.598003 56.057304 31 2.425318 L39 16.994211 46.505466 37 2.214019 I212 16.612061 96.245361 17 2.286034 L249 16.053497 89.531204 18 2.451091 N256 11.962807 74.001396 16 3.3658 V201 8.799002 32.334354 27 2.991305 F259 5.985607 52.407433 11 2.975162 Q204 3.71328 110.697441 3 3.752723 K210 2.756088 110.717255 2 3.567066 G202 1.427063 55.378868 3 4.671277
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Figure A5: Conservation of the ‘Proline+2’ ER export signal in invertebrates.
Fusion of the 1–35 N-terminal amino acids of human NUCB1, including both the signal
peptide (aa1–26) and the Proline residue at the +2 position (Pro+2) from the signal peptide
cleavage site (Pro+2), with enhanced green fluorescent protein (GFP), has been shown to be sufficient for localization in the Golgi, in HT1080 cells in culture (Tsukumo et al. 2009).
Single amino acid mutations of Pro+2 also resulted in defective export from the ER without
affecting the protein maturation process, in these experiments. Pro+2 was further observed in
this work to be important for the GFP fusion protein to concentrate at a transport vesicle
formation site within the ER, often termed the ER exit site. This residue has thus been
identified as a key determinant of NUCB1 protein export from the ER and subsequent
transport to the Golgi (Tsukumo et al. 2009).
The ‘Pro+2’ signal in NUCB1 is shownDraft (blue box with ‘P’ marked in red), as the second amino acid from the signal peptide cleavage site (downward pointing arrow) as being conserved from worms to humans, including dmNUCB1 and homologs from 4 other
Drosophila species.
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Figure A6: Appreciable conservation of the Leu/Ile-Rich Golgi Retention Sequence
(GRS) motif in invertebrates.
A deletion mutant of human NUCB2 or NEFA lacking amino acids 25-170 from the N-
terminal region, which are rich in leucine and isoleucine (hence denoted as the ‘Leu/Ile-rich’
region) has been demonstrated to not be retained in the Golgi, after cycloheximide treatment,
in HeLa cells in culture (Nesselhut et al. 2001). This region has therefore been designated as
a Golgi Retention Sequence (GRS). Human NUCB1 is 96% similar to its ortholog, NUCB2,
across this region. The 54aa-long core element of the GRS is shown to be appreciably
conserved from worms to humans and in dmNUCB1 (83% similarity to human NUCB1), as
also across all 4 other Drosophila species analyzed (80%, 87%, 87% and 85% similarity to
human NUCB1 for D.simulans, D.mojavensis, D.virilis and D.grimshawi, respectively). Draft
Figure A7: Conservation of the myristoylation / palmitoylation-based membrane lipid-
anchoring motif in Gαi/o invertebrate subunits.
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The amino-terminal 1-27aa stretch from rat Gαi3 has been implicated in membrane lipid anchoring (Lin et al. 1998). This region is substantially conserved from worm to human Gα subunits. All 5 Drosophila Gαi subunits are uniformly 82% similar to human Gαi1. All 5
Drosophila Gαoa subunits are 85% similar and All 5 Drosophila Gαob subunits are 92% similar to human Gαoa, respectively. The C.elegans GOA-1 subunit is 85% similar to human
Gαoa.
The G2 Glycine (myristoylation-related) and C3 Cysteine (palmitoylation-related) residues are shown to be identically and uniformly conserved with respect to Gαi from all 5
Drosophila species examined, Gαoa and Gαob isoforms from 4 different Drosophila species
(including D.melanogaster), Gαo from D.grimshawi and GOA-1 (Gαi/0) from C.elegans.
The two Gαo isoforms are seen to differDraft only across this N-terminal 27aa stretch, in all 5
Drosophila species. They are otherwise identical to each other, in primary amino acid sequence.
[Figure overleaf]
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Figure A7
Draft
Addendum:
Apart from the bioinformatics analyses shown here, it worth noting that:
1) The GBA motif has additionally been shown to be conserved elsewhere (Garcia-Marcos et
al. 2011) in Ornithorhynchus anatinus, Gallus gallus, Danio rerio, Tetraodon nigroviridis,
Ciona intestinalis and Nemostella vectensis.
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2) The entire GRS motif has been aditionally been shown to be conserved in the vertebrates
Mus musculus, Bos taurus, Xenopus laevis, Danio rerio and the insect Spodoptera frugiperada (Nesselhut et al. 2001).
3) cDNA clones sequence information from Spodoptera frugiperada (in turn, isolated via initial information based on an antigen deriving from a mouse monoclonal antibody raised against Golgi fractions from Sf21 insect cells) reveals the conservation of the Pro+2 signal and GBA motif (Kawano et al. 2000). In related electron microscopic studies from this work, insect NUCB1 was localized to the cis-Golgi cisternae and networks, in a pattern that closely parallels that of mammalian cells.
References
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List of Figure / Table Captions
Figure A1: The motif / domain-rich modularity of NUCB1 is mostly conserved across evolution from worms to humans, including 5 different Drosophila species.
Figure A2: The ‘PVQ5 repeat’ nomenclature proposed earlier for D.melanogaster NUCB1 inaccurately reflects the content of this region when assessed across 6 different invertebrate species.
Table AT1
The Variable Repeat Region differs considerably across invertebrate NUCB1 homologs, with respect to multiple criteria examined.
Figure A3: EF-hand 1 (EF1) and the proposed EF-hand 3 (EF3) are more stringently conserved in NUCB1 orthologs, than EF-handDraft 2 (EF2).
Figure A4(a): Near-complete conservation of residues crucial for functional linkage with
NUCB1, in invertebrate Gαi or Gαo subunits.
Figure A4(b): Significant conservation of multiple binding determinants in the GBA motif core of invertebrate NUCB1.
Table AT2: Conformational modeling of dmNUCB1 confirms the structural conservation of residues critical for its interaction with Gα subunits.
Figure A5: Conservation of the ‘Proline+2’ ER export signal in invertebrates.
Figure A6: Appreciable conservation of the Leu/Ile-Rich Golgi Retention Sequence (GRS) motif in invertebrates.
Figure A7: Conservation of the myristoylation / palmitoylation-based membrane lipid- anchoring motif in Gαi/o invertebrate subunits.
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