doi:10.1016/j.jmb.2010.01.009 J. Mol. Biol. (2010) 397,69–88

Available online at www.sciencedirect.com

RAB6C Is a Retrogene that Encodes a Centrosomal Involved in Cell Cycle Progression

Joanne Young1, Julie Ménétrey2 and Bruno Goud1⁎

1Molecular Mechanisms of Rab-GTPases are key regulators of membrane transport, and growing Intracellular Transport, CNRS, evidence indicates that their expression levels are altered in certain human UMR144, Institut Curie, malignancies, including cancer. Rab6C, a newly identified Rab6 subfamily 26 rue d'Ulm, 75248 Paris member, has attracted recent attention because its reduced expression Cedex 05, France might confer a selective advantage to drug-resistant breast cancer cells. Here, we report that RAB6C is a primate-specific retrogene derived from a 2Structural Motility, CNRS, RAB6A′ transcript. RAB6C is transcribed in a limited number of human UMR144, Institut Curie, tissues including brain, testis, prostate, and breast. Endogenous Rab6C is 26 rue d'Ulm, 75248 Paris considerably less abundant and has a much shorter half-life than Rab6A′. Cedex 05, France Comparison of the GTP-binding motifs of Rab6C and Rab6A′, homology Received 28 August 2009; modeling, and GTP-blot overlay assays indicate that amino acid changes in received in revised form Rab6C have greatly reduced its GTP-binding affinity. Instead, the 8 December 2009; noncanonical GTP-binding domain of Rab6C mediates localization of the accepted 5 January 2010 protein to the centrosome. Overexpression of Rab6C results in G1 arrest, Available online and its specific depletion generates tetraploid cells with supernumerary 11 January 2010 centrosomes, revealing a role of Rab6C in events related to the centrosome and cell cycle progression. Thus, RAB6C is a rare example of a recently emerged retrogene that has acquired the status of a new , encoding a functional protein with altered characteristics compared to Rab6A′. © 2010 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: centrosome; retrogene; Rab6A′; Rab6C; WTH3

Introduction RAB6B on 3 is a gene duplication of RAB6A/A′ not subject to splicing.2,3 Rab6A, 6A′,and Rab GTPases constitute the largest branch of the 6B localize at the late Golgi cisternae and trans-Golgi Ras superfamily and are involved in regulating network (TGN), as well as itinerant tubulovesicular intracellular protein trafficking pathways.1 Ypt6/ carriers that move along microtubules mainly in the 4–6 ′ RAB6 is one of five ancestral RAB conserved plus-end direction. Rab6A appears to be the from yeast to humans. During evolution, RAB6 closest functional equivalent to yeast homolog Ypt6, expanded by gene duplication and alternative splic- and like in budding yeast, depletion or overexpres- sion of mutant forms of Rab6A′ affects several ing, resulting in the expression of three RAB6 isoforms 7 in Homo sapiens. The primordial RAB6A′/A gene is trafficking routes that intersect the Golgi. This encoded on human and generates includes early endosome-to-TGN retrieval pathways two spliced variants, RAB6A′ and RAB6A,while that carry such as TGN46, mannose 6- phosphate receptors, or internalized glycolipid- bound toxins;8–13 recycling of Golgi-resident enzymes *Corresponding author. E-mail address: to the ;14–17 and efficient [email protected]. delivery and exocytosis of cargo to the plasma Abbreviations used: GFP, green fluorescent protein; membrane.5 In each pathway, the function of Rab6A′ TGN, trans-Golgi network; ORF, open reading frame; may be manifold, acting in conjunction with other OWM, Old World monkey; BrdU, bromodeoxyuridine; molecular components to dock incoming vesicles,18 FRAP, fluorescence recovery after photobleaching; HA, organize subdomains for membrane budding/ hemagglutinin epitope; siRNA, short interfering RNA; fission,19 or recruit/activate molecular motors.5,20,21 GDI, guanine dissociation inhibitor; GST, glutathione S- The diverse and dynamic functions of the Rab6 transferase; UTR, untranslated region; HRP, horseradish isoforms are reflected in the identification of more peroxidase; PBS, phosphate-buffered saline; NCBI, than 15 interacting effector proteins that include National Center for Biotechnology Information. motors, motor adapters, tethers, and structural

0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved. 70 A Retrogene with Functional Status

– proteins.8,9,20,22 25 Some effort has been made to Here, we completely reassess RAB6C in light of our understand the specific roles of the structurally and finding that it originated by retrotransposition, a functionally related Rab6 subfamily isoforms. Rab6A mechanism whereby mRNA is reverse transcribed and Rab6A′ are the most ubiquitously and abun- and randomly integrated into the genome.35 Most dantly expressed members, differing in just three retrotransposition events result in inactive pseudo- amino acids, and have similar GTP-binding genes and are a problematic source of experimental properties.2 How redundant or essential the individ- artifacts.36 Rarely though, a new retrogene encoding a ual activities of Rab6A and Rab6A′ are to the protein with modified or novel functions can emerge. membrane-trafficking pathways mentioned above is Here, we demonstrate that retrogene RAB6C is such still under investigation.26 The particular function of an example, encoding a biologically relevant protein the more divergent Rab6B isoform is easier to infer, it that is, however, completely unconventional as a Rab. being selectively expressed in neuronal cells, and is Instead, primate-restricted Rab6C is a centrosomal presumed to have a novel spatiotemporal role protein with a role in processes related to regulation necessary for the complex trafficking pathways that of centrosome duplication and cell cycle progression. operate in specialized cell types.3 Finally, in addition to promoting membrane trafficking during inter- Results phase, at least one of the Rab6 isoforms plays a role during mitosis. Thus, Rab6A′ is required for proper inactivation of the Mad2 spindle assembly check- RAB6C is a hominoid-specific intronless gene point, and unexpectedly, this second function requires derived from RAB6A′ mRNA neither prenylation nor GTP hydrolysis by Rab6A′.27 Recently, two groups discovered a fourth Rab6 Within the , RAB6C (GenBank isoform denoted as WTH3 or RAB6C. WTH3 was accession 84084) maps to chromosome 2q21.1 isolated as a differentially methylated DNA frag- (Supplementary Fig. S1a). Given its single-exon ment, and RAB6C was identified as a novel cDNA in structure and that it shares highest identity to the context of the LIFEdb project.28,29 Rab6C shows RAB6A′ transcript (97%), RAB6C was likely gener- highest identity to Rab6A′ (91%) but possesses an ated by retrotransposition of a fully processed extra carboxy-terminal extension of 46 amino acids RAB6A′ mRNA. A second RAB6C-like intronless after the hypervariable region, suggestive of unique gene (GenBank accession 150786) exists approxi- functional properties. In accordance with this idea, mately 1.38 Mb downstream from RAB6C in an green fluorescent protein (GFP)-tagged Rab6C did inverted orientation (Supplementary Fig. S1a). The not show subcellular localization at the Golgi or sequences of RAB6C and RAB6C-like are almost delay transport of the secretory cargo VSV-G to the identical over 21,000–24,000 bp of flanking region, plasma membrane like Rab6A′.30 Instead, Rab6C indicating that one of the copies is the result of an was found to have a nucleo-cytoplasmic intrachromosomal segmental duplication rather distribution.31 The promoter region of RAB6C is than a second retrotransposition event. associated with a CpG island that is partially To determine the timing of the retrotransposition methylated in primary cells isolated from effusions event, we searched for intronless RAB6C orthologues of breast cancer patients and hypermethylated in in other species. RAB6C was absent from bovine and drug-resistant cell lines but not in paired drug- rodent genomes, suggesting that it was acquired sensitive cell lines.31,32 Hypermethylation of RAB6C during anthropoid evolution. Further analysis results in downregulation of its mRNA levels, and revealed the occurrence of RAB6C in four apes addition of histone deacetylase and methyltransfer- (chimpanzee, gorilla, orangutan, and gibbon) but ase inhibitors reversed epigenetic silencing of not in any of the available Old World monkey (OWM) RAB6C.32 Stable re-expression of RAB6C in a genomes (Supplementary Table S1). To verify that multidrug-resistant uterine sarcoma cell line RAB6C is indeed absent from OWM, we screened resulted in 8–35 times increased sensitivity to the genomic DNA isolated from rhesus macaque and chemotherapeutic agent doxorubicin.29 Conversely, Vero and COS-7 cell lines originated from green reduction of RAB6C expression in HEK293 cells monkey. A chimpanzee genomic sample served as a using an RNAi strategy made them 3–7 times more control for the existence of the RAB6C gene. Primers tolerant to doxorubicin than wild-type HEK293 designed against conserved sequences of RAB6C only cells.33 More recently, RAB6C was found to harbor amplified a PCR product of the expected size of an unusual but functional p53-binding motif within intronless RAB6C in chimpanzee (Supplementary Fig. its promoter region. Overexpression of p53 in- S1b). We can thus estimate that the retrotransposition creased levels of RAB6C mRNA in HeLa cells but event giving rise to RAB6C occurred after the OWM– not in drug-resistant MCF-7 cells, where promoter hominoid split and before separation of the lesser ape methylation abrogated transactivation by p53. Fur- and other hominoid lineages, between 21 and 25 thermore, overexpression of Rab6C in HEK293 cells MYrs ago (Fig. 1a).37 Inspection of the putative stimulated apoptosis.33,34 These results lead to the translation products corresponding to RAB6C and current hypothesis that RAB6C is a candidate tumor RAB6C-like revealed preservation of the open reading suppressor gene encoding a pro-apoptotic factor frame (ORF) in human, chimpanzee, gorilla, and and necessarily downregulated during establish- orangutan. In contrast, multiple stop codons in the ment of drug resistance in order to avoid cell death. gibbon sequence prevent an elongated ORF, A Retrogene with Functional Status 71

Fig. 1. Estimation of the age of RAB6C insertion and analysis of RAB6C expression. (a) RAB6A′ mRNA integrated in a common ancestor of apes and humans and was duplicated by chromosomal segmentation. Nonfunctional copies of RAB6C and RAB6C-like are shown in gray although the inactivating stop codons are recent and not ancestral events. Yellow indicates the C-terminal extension of Rab6C. Numbers refer to approximate divergence times in millions of years. (b) Expression profile of RAB6C, RAB6A′, and RAB6B in 20 different human tissues. RAB6C (550 bp) shows highest expression in brain, prostate, and testis. Constitutive levels of expression are observed in all tissues for RAB6A′ (340 bp) while RAB6B (597 bp) is predominantly expressed in brain. suggesting pseudogenization in the modern-day implies a tissue-specific role and was distinct from species (Supplementary Fig. S2 and data not shown). that of ubiquitously expressed RAB6A′ (and RAB6A; data not shown) and the neuronal-specific isoform RAB6C is transcribed in a tissue-specific manner RAB6B (Fig. 1b). We next investigated in which cell lines RAB6C is expressed. Out of 17 cell lines, Previous studies29 have suggested that RAB6C is RAB6C transcript was detected in only HEK293, ubiquitously expressed like RAB6A′; however, the HEK293T, LNCaP, MCF-7, T-47D, and EVSA-T cells authors were unaware that RAB6C is an intronless (Fig. 2a and Supplementary Table S2). Although gene, which complicates analysis of RNA RAB6C is expressed in normal human breast tissue, expression.38 To confirm the existence of RAB6C half of the breast cancer cell lines we examined did transcript, we used specific primers that discrimi- not express RAB6C, suggesting frequent down- nate between nearly identical RAB6C and RAB6A′ regulation of RAB6C expression. mRNA sequences (Supplementary Fig. S1c), and RT-PCR analysis was carried out on a panel of Detection of endogenous Rab6C RNAs isolated from 20 human normal tissues. Highest levels of RAB6C were found in fetal and To evaluate if RAB6C is a protein-coding retro- adult brain, prostate, testis, and spinal cord and gene, we generated a polyclonal antibody using full- were undetectable in adrenal gland, skeletal muscle, length recombinant Rab6C purified as a His tag bone marrow, fetal, and adult liver, heart, salivary fusion (see Materials and Methods). Given the high gland, and trachea. The expression profile of RAB6C primary sequence similarity between Rab6C and 72 A Retrogene with Functional Status

Fig. 2. Expression of RAB6C mRNA and Rab6C protein in human cell lines. (a) RAB6C (550 bp) was amplified specifically upon addition of AMV reverse transcriptase in certain cell lines. Internal control GAPDH was detected in all cell lines (1000 bp), confirming the quality and integrity of the RNA. Further controls consist of plasmids containing cDNA sequences of RAB6C, RAB6A′,orRAB6B to demonstrate the specificity of primers. Note that RAB6C is expressed in normal breast and prostate tissue but not all breast and prostate cell lines express RAB6C. (b) Rab6C (30 kDa; arrowhead) is expressed in certain cell lines and at much lower levels than Rab6A/A′. To detect Rab6C, we subjected 1 mg of protein from each cell line to immunoprecipitation followed by immunoblotting (IP+WB) with anti-Rab6C polyclonal antibody. With the same antibody, Rab6A/A′ (25 kDa) isoforms can be detected in all cell lines simply by immunoblotting (WB) 25 μg of protein lysate. Calnexin (90 kDa) is shown as a loading control. Asterisks refer to nonspecific bands.

Rab6A/A′, it was possible that the resulting anti- inhibit protein synthesis, Rab6C degraded rapidly Rab6C antiserum would exhibit cross-reactivity. such that within 1 h of treatment, less than 50% of This was confirmed by Western blotting against the protein remained (n=3 experiments; Supple- lysates of cells overexpressing GFP-Rab6C and GFP- mentary Fig. S3c). Ectopically expressed GFP-Rab6C Rab6A′; however, the antibody clearly possessed also decayed rapidly with kinetics similar to that of higher affinity for GFP-Rab6C (Supplementary Fig. endogenous Rab6C (Supplementary Fig. S3c). In S3a). Anti-Rab6C also detected endogenous Rab6A/ contrast, levels of Rab6A/A′ and GFP-Rab6A′ A′ at 25 kDa in all cell lines tested upon immuno- stayed relatively constant over 5 h of cycloheximide blotting of 40 μg of cell lysate (Fig. 2b), but no band treatment, reflecting a comparatively longer half-life corresponding to endogenous Rab6C at 30 kDa and increased protein stability (Supplementary Fig. could be observed (data not shown). Detection of S3c). Addition of lysosomal and proteasome inhibi- Rab6C was eventually achieved by using a more tors did not increase levels of endogenous Rab6C, sensitive immunoprecipitation/immunoblot proce- indicating that these pathways are not implicated in dure from 3 mg of cell lysate, indicating that steady- Rab6C degradation (Supplementary Fig. S3d). state levels of Rab6C are significantly lower than Rab6A/A′ (Fig. 2b). Using this method, we detected Rab6C is an inefficient GTP-binding protein Rab6C exclusively in those cell lines previously compared to Rab6A′ shown to express RAB6C by RT-PCR analysis (Fig. 2a). Furthermore, the 30-kDa band attributed to Amino acid alignment of human Rab6C and endogenous Rab6C did not appear when lysates Rab6A′ revealed that Rab6C harbors important were immunoprecipitated in the absence of anti- substitutions in four out of five elements (G1–G5) Rab6C antibody (Supplementary Fig. S3b). critical for GTP binding and hydrolysis (Table 1). We explored if posttranslational mechanisms are The most striking change is a noncanonical alanine responsible for the low basal level of Rab6C. After substituting the strictly conserved glycine residue of treatment of HEK293 cells with cycloheximide to the G1 motif (P-loop) that interacts with the A Retrogene with Functional Status 73

Table 1. Comparison of the GTP-binding motifs of Rab6C orthologues and human Rab6A′

Isoform Species G1 G2 G3 G4 G5 Rab6C Human GEQSVAKT I DTAGQ NRTD ETRAK Chimpanzee GEQSVAKT T DTAGQ NRTD ETRAK Gorilla GEQSVAKT I DTAGQ NRTD ETRAK Orangutan GEQSVAKT T DTAGQ NKTD ETSAK Rab6A′ Human GEQSVGKT T DTAGQ NKTD ETSAK Canonical sequence GxxxxGKT T DxxGQ NKxD ETSAK X denotes any amino acid. Letters in bold italics denote changes from the consensus.

Fig. 3. Molecular modeling of human Rab6C structure and GTP-binding analysis. (a) The model uses the structure of the G-domain of Rab6A′ ( ID: 1YZQ39) as a template. An overall view of human Rab6C (residues 14– 175) is shown in blue with the switch regions indicated in light blue and sequence differences with Rab6A′ indicated in red with the side chains shown in stick representation (left panel). A detailed view of the nucleotide binding site of human Rab6C is shown (right panel) with differences from Rab6A′ indicated in green. A sequence difference at position 25 of the P-loop in Rab6C should induce steric hindrances with the nucleotide and/or with the conserved Asn127 residue that is critical for binding and specific recognition of the guanine part of the nucleotide. (b) Coomassie staining and [α-32P]GTP overlay blot assay of purified GST-Rab fusion proteins. GST serves as a negative control for background levels of [α-32P] GTP. One microgram of purified protein was separated by SDS-PAGE and stained with Coomassie blue or transferred to nitrocellulose membranes. The nitrocellulose blots were incubated with [α-32P]GTP, washed, and exposed to x-ray film. Purified GST-Rab6C and GST-Rab6C mutants are more prone to degradation than GST-Rab6A′, giving rise to several smaller molecular weight products. Arrowheads indicate full-length GST fusion proteins. 74 A Retrogene with Functional Status phosphate β of the nucleotide (GxxxxGKT sequence sequences, indicating that it appeared soon after in Rab6A′ is replaced by GxxxxAKT in Rab6C). The insertion of ancestral RAB6C into the genome Gly25Ala replacement exists in all hominoid Rab6C (Supplementary Fig. S2).

Fig. 4 (legend on next page) A Retrogene with Functional Status 75

We generated a homology model using the crystal order to conform to the canonical P-loop consensus structure of human Rab6A′ G-domain.39 Based on as found in Rab6A′. However, the restored P-loop the computational model, the Gly25Ala replacement motif in GST-Rab6CA25G did not result in detectable is predicted to affect the overall conformation of the GTP binding (lane 10). Rab6C mutants with restored P-loop in Rab6C (Fig. 3a). More specifically, the P-loop and G2 motifs (GST-Rab6CA25G/I45T; lane 14) Gly25Ala replacement should provoke steric hin- or, ultimately, all four motifs involved in GTP drances with the nucleotide base and the conserved binding (data not shown) also failed to bind Asn126 from the G4 motif (NKTD), reducing the detectable levels of GTP. This suggests that addi- ability of Rab6C to interact and stabilize the tional substitutions adjacent to the G1–G5 motifs in nucleotide, whether GDP or GTP (Fig. 3a). Further- Rab6C affect the flexibility of the nucleotide binding more, Lys127 from the G4 motif that makes pocket and impact upon GTP binding. Lastly, important hydrophobic interactions with the gua- introduction of the P-loop of Rab6C into Rab6A′ ′ nine base is replaced in Rab6C by an arginine abolished the ability of GST-Rab6A G25A to bind residue (except in orangutan; see Table 1 and GTP (lane 10), confirming the importance of Gly25 Supplementary Fig. S2). This difference is also within the P-loop for competent GTP binding. These expected to affect nucleotide binding, since an experimental results corroborate the computational arginine residue with its longer side chain cannot model prediction that sequence changes accumulat- strictly mimic the lysine residue that extends to ed in Rab6C have strongly reduced its ability to bind make hydrogen bonds with the main chain of the P- GTP compared to Rab6A′. loop (Fig. 3a). Also of note is the replacement in Rab6C of the conserved threonine of the G2 motif, Rab6C associates with the centrosome crucial for switch I to sense the presence of the gamma phosphate of GTP and subsequently trigger Since the anti-Rab6C antibody simultaneously the conformational change required for effector detected the more abundant Rab6A/A′ isoforms recognition. This is notexpectedtooccurin upon immunostaining of fixed cells (data not Rab6C, which has an isoleucine residue at this shown), we were obliged to study Rab6C fused to position, further indicating that Rab6C is an atypical a fluorescent or epitope tags. GFP-Rab6C transiently switch protein (Fig. 3a). This sequence difference expressed in HeLa cells showed a diffuse cytosolic- exists in human and gorilla Rab6C, whereas like pattern but no association with the Golgi, as chimpanzee and orangutan possess the conserved previously reported.31 Moderate levels of GFP- threonine residue (Table 1; Supplementary Fig. S2), Rab6C expression did not affect overall Golgi suggesting recent loss of the switch I ability to sense structure as labeled by GM130, a Golgi matrix GTP. protein, or the localization or levels of endogenous Rab6C thus lacks a canonical P-loop and other Rab6A/A′ associated with the Golgi (Fig. 4A). More consensus GTP-binding motifs and is not expected careful microscopic examination revealed that in 20– to be a conventional GTP-binding protein. Surpris- 30% of transfected cells, GFP-Rab6C concentrated at ingly, however, Rab6C was previously reported to one or two pairs of dots confirmed to be centrioles exhibit GTP-binding affinity comparable to that of by double immunostaining with antibodies directed Rab6A′.31 We reexamined the ability of Rab6C to against known centrosomal markers (Fig. 4A, insets, bind GTP using a [α-32P]GTP overlay blot assay and and Fig. 4B). The presence of GFP-Rab6C at the purified glutathione S-transferase (GST)-tagged centrosome in a subset of the transfected cell fusion proteins (see Materials and Methods). No population suggests that it may be associated with GTP binding was detected for GST-Rab6C (Fig. 3b; the centrosome spatiotemporally, like certain other lanes 6) even upon removal of its 46-amino-acid C- centrosomal components.40 Other tagged versions – terminal extension (GST-Rab6C1 208; lane 7). Under of Rab6C and the chimpanzee GFP-Rab6C ortholo- the same conditions, other Rab proteins including gue associated with the centrosome and also in GST-Rab6A′ all bound GTP (lanes 2–5). Moreover, various different human cell lines (Supplementary GST-Rab6A′, when fused to the C-terminal exten- Fig. S4a). Pre-extraction of cells with detergent sion of Rab6C (GST-Rab6A′+ext; lane 9), bound before fixation showed that centrosomal located GTP, indicating that the C-terminal extension is not GFP-Rab6C resists detergent, indicating a strong responsible for the lack of GTP binding. We replaced association (Supplementary Fig. S4b). After nocoda- the Ala25 residue in Rab6C by a glycine residue in zole treatment to depolymerize microtubules, GFP-

Fig. 4. Localization of GFP-Rab6C at the centrosome and analysis of Rab6C dynamics. (A) Fluorescence images of HeLa cells transiently expressing GFP-Rab6C for 20 h and stained with anti-GM130 (upper panel) or anti-Rab6A/A′ (lower panel) antibodies, in order to visualize the Golgi apparatus. Insets show higher magnification of GFP-Rab6C enriched in the centrosomal region. (B) To demonstrate centrosomal localization, we costained cells with antibodies against (a) γ-tubulin, (b) ninein, or (c) AKAP450. Arrows indicate the centrosome. (C) FRAP analysis of centrosomal-associated mCherry-Rab6C expressed in a stable HeLa Centrin1-GFP cell line to track centrosomal position. The average recovery profiles from a total of eight cells are presented. Error bars indicate standard deviation. Rate of recovery was so slow that it could not be accurately calculated within the time scale of 250 s. Still images from a longer-term 20-min postbleaching experiment of mCherry- Rab6C are shown in the right panel. Insets show the unbleached centrosome region indicated by an arrow before (prebleach), immediately after (0), and 5 and 20 min after photobleaching. The scale bar represents 10 μm in each case. 76 A Retrogene with Functional Status

Rab6C remained at the centrosome, indicating that plex for lipid modification. Further deletion analysis GFP-Rab6C is not simply a microtubule minus-end- identified the G-domain of Rab6C, covering amino associated protein but genuinely associates with the acids 1–174 as the minimal centrosomal binding – centrosome (Supplementary Fig. S4c). Overexpres- domain (GFP-Rab6C1 174; Fig. 5b). sion of GFP-Rab6C had no noticeable effect on To understand if Rab6C localization at the microtubule organization, and in nocodazole wash- centrosome is a specific feature of Rab6C, we out experiments, the rate of microtubule reforma- constructed plasmids expressing hybrid polypep- tion was comparable in GFP-Rab6C-expressing and tides of GFP-Rab6A′ and GFP-Rab11A fused to the nontransfected cells (data not shown). Substitutions unique 46-amino-acid C-terminal extension of that lock the close homologue Rab6A′ into a Rab6C. As shown in Fig. 5c (upper panel), the constitutively GTP-bound active (Q72L) or a GDP- addition of the extension drastically altered the bound inactive (T27N) state were introduced into subcellular localization of GFP-Rab6A′ and GFP- Rab6C to assess the importance of any putative Rab11, normally situated at the Golgi and peri- GDP/GTP binding and hydrolysis activity of Rab6C nuclear recycling endosomes, respectively (Fig. 5c, for centrosomal localization.15 However, neither lower panel), and likely due to the absence of prenyl GFP-Rab6CQ72L nor GFP-Rab6CT27N showed any conjugation of GFP-Rab6A+ext and GFP-Rab11 qualitative or quantitative difference in centrosomal +ext (Supplementary Fig. S5c; data not shown). association compared to wild-type Rab6C, provid- The hybrid polypeptides GFP-Rab6A′ +ext and ing further indication that Rab6C is not a conven- GFP-Rab11+ext appeared as multiple dots of tional Rab protein (data not shown). various sizes scattered throughout the cytoplasm To gain insight into the dynamics of Rab6C, we (Fig. 5c, upper panel). These punctate structures did used fluorescence recovery after photobleaching not overlap with GM130, transferrin receptor, early- (FRAP). mCherry-Rab6C was transiently expressed endosomal antigen-1, or lysosomal associated mem- in a HeLa Centrin1-GFP stable cell line,41 allowing brane protein-1, indicating that they are not derived dual imaging of unbleached Centrin1-GFP to track from Golgi or endosomal membranes (data not the centrosome and simultaneous measurement of shown). Although some of the dots congregated mCherry-Rab6C bleach and recovery. After photo- close to the centrosome, they failed to colocalize bleaching, virtually no recovery of mCherry-Rab6C with the centrosome itself as revealed by costaining fluorescence at the centrosome was observed over a with γ-tubulin (Fig. 5c, insets). The dotty aspect of 250-s period (Fig. 4B, left panel). Some recovery was GFP-Rab6A+ext and GFP-Rab11+ext is reminis- obtained by 20 min postbleach (Fig. 4B, right panel). cent of multiple small aggregates that are trans- TheslowfluorescencerecoveryofRab6Cwas ported in a microtubule-dependent manner towards comparable to that observed upon photobleaching the microtubule-organizing center, eventually form- of the core centrosomal component Centrin1-GFP ing a large central aggresome.42 We did not pursue (Supplementary Fig. S5a). In contrast, the recovery this observation further since the hybrid polypep- half-life of the cytoplasmic GFP-Rab6C pool was tides are chimeras that perhaps give rise to rapid, the mobile fraction recovering to 72% with a misfolded proteins and a protective cellular re- ∼ t1/2 5s(Supplementary Fig. S5b). The results sponse. The inability of the chimeric Rabs to indicate that ectopically expressed Rab6C is stable associate with the centrosome strengthens the at the centrosome. notion that Rab6C location at the centrosome is regulated and functionally relevant. The unconventional G-domain of Rab6C is The unique C-terminal extension of Rab6C does sufficient for its centrosomal localization not contain a centrosomal targeting sequence and has no motifs or homology to any other protein. To To map the region within Rab6C required for understand the origin of the novel sequence, we centrosome association, we analyzed the localiza- compared the RAB6C nucleotide sequence that tion of various deletion mutants (Fig. 5a). Although encodes the C-terminal amino acids 209–254 to the Rab6C possesses a CxC prenylation motif at the end 3′-untranslated region (UTR) sequence of RAB6A′ of its hypervariable region, the additional extension (Supplementary Fig. S5d). Both nucleotide prevents prenylation (Supplementary Fig. S5c). In sequences are almost identical and demonstrate – the truncated construct GFP-Rab6C1 208, the CxC that the extension resulted from a point mutation in prenylation motif at the end of the hypervariable the stop codon of an ancestral RAB6C gene (627ANC, region is potentially available for prenylation and p.X209Y), allowing the reading frame to continue we predicted that it might localize at the Golgi until the next stop codon downstream. This point – complex like Rab6A′. GFP-Rab6C1 208, however, mutation is present in all RAB6C orthologues, still associated with the centrosome only (Fig. 5B) indicating that it occurred soon after the initial and a Triton X-114 partitioning assay showed that retrotransposition event (Supplementary Fig. S2). – GFP-Rab6C1 208 is not prenylated (Supplementary Fig. S5c). Presumably, even when tailored to termi- Overexpression of Rab6C promotes cell cycle nate with a CxC targeting motif, the 22-amino-acid arrest leading to apoptosis – substitutions that differentiate Rab6C1 208 from Rab6A′ have affected its ability to interact with the Many centrosomal-associated proteins exhibit Rab escort protein:geranylgeranyl transferase com- variations in expression levels or activity during A Retrogene with Functional Status 77

Fig. 5. Identification of the domain mediating Rab6C localization at centrosomes. (a) Schematic representation of full- length Rab6C protein. Indicated are the N-terminal unconventional G-domain of Rab6C and the hypervariable region ending in a prenylation motif (CxC) in blue. The cysteine targeting sequence is not prenylated in Rab6C due to the presence of an extending 46-residue C-terminal extension, highlighted in yellow. Truncation constructs used in the study are shown below. (b) Centrosomal association of Rab6C occurs through its N-terminal atypical G-domain. HeLa transfected with constructs encoding GFP-tagged deletion mutants of Rab6C (green) were fixed after 24 h and immunostained with γ-tubulin (red). The region of the centrosome (arrow) is detailed in insets. (c) Subcellular localization of GFP-Rab6A′ and GFP-Rab11A fused to the C-terminal extension sequence of Rab6C (upper panel). The hybrid polypeptides do not colocalize with the centrosome labeled by γ-tubulin (red) as detailed in the insets. Localization of wild-type GFP-Rab6A′ (Golgi) and GFP-Rab11A (perinuclear) are shown in the lower panels. Scale bars represent 10 μm. 78 A Retrogene with Functional Status the cell cycle. Using MCF-7 cells synchronized in decreased to 35±3% of the levels found during G1/G0, S, S/G2, or M phase, we observed that interphase (n=3 experiments; Fig. 6a). In contrast, levels of Rab6C in M phase were significantly levels of Rab6A/A′ and β-tubulin were continuous

Fig. 6 (legend on next page) A Retrogene with Functional Status 79

Fig. 7. Overexpression of Rab6C results in cell cycle arrest before the G1/S transition and accumulation of cells in G1 phase. (a) Twenty-four hours after transfection of HeLa cells with constructs encoding various GFP fusion proteins, BrdU was added to the media for 10 min. Cells were fixed and stained with anti-GFP, anti-BrdU antibodies, and 4′,6-diamidino- 2-phenylindole. The percentage of BrdU-positive cells is presented. More than 100 cells were scored for each construct, and the results are an average of experiments performed at least twice. Error bars indicate standard deviation. (b) DNA histograms of cells expressing H2B-GFP or GFP-Rab6C after 24 h of expression. Cells were trypsinized, followed by fixation and propidium iodide staining. GFP fluorescent cells were sorted and analyzed for DNA content and cell cycle profile by flow cytometry. Event count (y-axis) versus DNA content (x-axis) of a single typical experiment is shown. The percentage of cells in the G1, S, and G2/M phases of the cell cycle was determined using ModFit and are expressed as means±SDs of three independent experiments. throughout the cell cycle. Synchronized HEK293 GFP as a cytological marker for centrosome matu- cells also showed Rab6C levels of 24±9% in mitosis ration and cell cycle progression, we observed that compared to interphase (n=3 experiments; Supple- mCherry-Rab6C can associate with both undupli- mentary Fig. S6a). This suggests that Rab6C is a cell- cated and duplicated centrosomes in some cells cycle-regulated protein. In addition, using Centrin1- (Fig. 6b).41 However, mitotic cells expressing

Fig. 6. Endogenous Rab6C levels are decreased in mitosis and cells ectopically expressing Rab6C fail to enter mitosis. (a) MCF-7 cells were nonsynchronized (NS) or synchronized at various stages of the cell cycle using double-thymidine block (S phase), double-thymidine and washout (G2 phase), or nocodazole treatment (M phase). Rab6C (arrow) was immunoprecipitated from cell lysates. Asterisks are nonspecific bands. In parallel, Rab6A/A′ and β-tubulin levels were monitored by immunoblotting. The efficiency of synchronization is indicated by the percentage of cells quantified in each stage of the cell cycle by flow cytometry. (b) Localization of mCherry-Rab6C (red) in HeLa cells stably expressing centriolar marker, Centrin1-GFP (green). Deconvolved images of a G1 phase cell with two centrioles (upper panel) and S/ G2 phase cell with four centrioles (lower panel). The scale bar represents 1 μm. (c) Transfected cell populations were analyzed in a time period between 24 and 50 h after transfection using time-lapse video microscopy. Pictures were taken every 15 min over a period of 26 h of which phase contrast and fluorescent images at every 6-h time point are presented here: 24, 30, 36, 42, and 48 h. Arrows follow individual transfected cells over the time course. Compared to mito-GFP- transfected cells (left panels) that divide several times over the time course of the experiment, GFP-Rab6C-transfected cells (right panels) arrest sometimes, followed by cell death (asterisks). The scale bar represents 10 μm. 80 A Retrogene with Functional Status mCherry-Rab6C were never found, indicating that To examine if ectopic expression of Rab6C indeed ectopic expression of Rab6C may affect cell cycle affects cell cycle progression, we followed cells progression. from 18 to 44 h post-transfection by time-lapse

Fig. 8 (legend on next page) A Retrogene with Functional Status 81 microscopy. During the 26 h of video acquisition, to inhibit Rab6C expression in HEK293 cells. Due to 95% of GFP-Rab6C-expressing cells (n=112 cells) the high sequence identity of RAB6C and RAB6A′ failed to enter mitosis (Fig. 6c and Supplementary transcripts, we found it technically impossible to Video S1, right panels), while 67% of cells trans- knock down RAB6C without affecting RAB6A′ fected with mito-GFP (n=61) divided at least once levels (see siRNAs 259, C6, 333 in Supplementary (Fig. 6c and Supplementary Video S1, left panels). In Table S3). We therefore established a stable HEK293 cells expressing GFP-Rab6C for a period of more cell line expressing a GFP-tagged siRNA-resistant than 12 h (n=70 cells), 67% were observed to die in a form of RAB6A′ (GFP-siResRab6A′)previously process that morphologically resembled apoptosis shown to compensate all functions of endogenous (see Supplementary Video S1, right panels). Under Rab6A′ after depletion of the latter by RNAi.12,27 the same conditions, only 13% of cells expressing With the use of this strategy, two independent mito-GFP for more than 12 h (n=58 cells) during siRNAs (named siRNA1 and siRNA2) were shown video acquisition died in this manner. These by immunoprecipitation to efficiently deplete en- observations suggest cells ectopically expressing dogenous Rab6C levels (Supplementary Fig. S6a). Rab6C cell cycle arrest, leading to subsequent At 48 and 72 h post-transfection of HEK293 GFP- apoptosis. To verify that cells expressing GFP- siResRab6A′ cells, siRNA1 decreased Rab6C to 8 Rab6C do not enter S phase, we incubated cells ±7% and 28±5% of that found in control-siRNA- with bromodeoxyuridine (BrdU). Quantification treated cells (n=3 experiments). siRNA2 was slight- showed an ∼80% decrease in the number of cells ly less efficient, reducing levels of endogenous that incorporate BrdU compared with controls (Fig. Rab6C to 36±9% at both time points (n=3 experi- 7a). Note that HA (hemagglutinin epitope)-Rab6C, ments). As expected, levels of endogenous Rab6A′ – – GFP-Rab6C1 208, GFP-Rab6C1 174, and Q72L and were similarly diminished, while GFP-siResRab6A′ T27N variants of GFP-Rab6C also prevented cells levels remained constant (Supplementary Fig. S6a). from entering S phase to the same extent as GFP- We noticed that at 72 and 96 h post-transfection Rab6C (Fig. 7a). Similar results were obtained in with siRNA1 and siRNA2, the number of cells/well other cell lines including HEK293, HCT116, and was 60% less than cells treated with control siRNA MCF-7 cells (data not shown). Finally, subsequent (Fig. 8a), indicating that downregulation of Rab6C FACS analysis confirmed an effect of Rab6C over- affects cell proliferation. HEK293 GFP-siResRab6A′ expression on cell cycle distribution (Fig. 7b). Thus, treated with siRNA2 were harvested at 72 h post- expression of GFP-Rab6C resulted in an increased siRNA transfection and analyzed by FACS to assess percentage of cells in the G1/G0 phase to 90.3± if knockdown of RAB6C induces cell cycle arrest 1.5%, whereas control-transfected cells accounted (Fig. 8b). siRNA2-treated cells showed a higher for 61.7±6.4% in the G1/G0 phase. The difference number of cells with 4N DNA content (19±2.6%) was repeatedly observed in three independent compared with controls (7±1.5%) and only a low experiments and was accompanied by a propor- number of cells in the sub-G1 fraction that represent tional decrease of cells in S and G2/M phases to 6± dead cells (n=3 experiments). An increase in the 1% and 3.7±1.2%, respectively, as compared to 30± number of cells with 4N DNA content may signify a 4.4% and 8.3±2.1% in control-transfected cells. Since redistribution of cells to the G2/M phase of the cell Rab6C-expressing cells were positive for the prolif- cycle or an aberrant G1 state with 4N DNA eration marker Ki67 (data not shown), these findings (tetraploidy) due to mitotic slippage or failure of suggest that Rab6C imposes a delay in cell cycle cytokinesis. Since, by immunostaining, there was no progression during G1. obvious increase in the number of mitotic or G2 phase cells compared to control-siRNA-treated samples (data not shown), the increase of cells with 4N Depletion of Rab6C induces tetraploidization content more likely corresponds to tetraploidization. and supernumerary centrosomes We evaluated whether silencing of Rab6C in HEK293 GFP-siResRab6A′ cells affects the number To assess if endogenous levels of Rab6C are of centrosomes per cell. At 72 and 96 h after important for cell cycle progression, we used transfection with siRNA1, an increase of cells with synthetic short interfering RNA (siRNA) duplexes N2 centrosomes was observed in the Rab6C-

Fig. 8. Depletion of Rab6C in HEK293 GFP-siResRab6A′ cells results in decreased growth rate, tetraploidy, and supernumerary centrosomes. (a) Representative growth curve showing that downregulation of Rab6C by two different siRNA sequences significantly reduces cell growth. Cell number count of HEK293 GFP-siResRab6A′ cells seeded in 12- well dishes at 125,000 cells/well and transfected 24 h later with control luciferase siRNA, siRNA1, or siRNA2. Cells were counted in duplicate for a period of 5 days after transfection. (b) Flow cytometry profiles of cells treated with control or siRNA2 for 72 h (N10,000 cells per trace) from a single typical experiment are shown. The percentage of G1, S, and G2 stage cells after each treatment is expressed as the mean±SDs of three independent experiments. (c) Immunofluorescence microscopy images of control and Rab6C-depleted HEK293 GFP-siResRab6A′ cells stained with anti-GMAP210 (green) to visualize the Golgi and anti-AKAP450 (red) to label centrosomes. Nuclei (blue) were stained with 4′,6-diamidino-2- phenylindole. Boxed areas are magnified in the insets. Arrowheads indicate binucleated cells. The scale bar represents 10 μm. (d) Quantification of HEK293 GFP-siResRab6A′ or HeLa cells with multiple centrosomes (N2 AKAP450-labeled dots per cell) following siRNA1 treatment for 72 or 96 h. Error bars indicate standard deviation. For each bar of the graph, nN1500 cells and the graph is representative of two experiments. 82 A Retrogene with Functional Status deficient population, many of which were binucle- protein compared to Rab6A′. Rab6C is highly ated (arrows in Fig. 8c). Note that as evaluated by unlikely to have the same GTP-binding properties GMAP210 staining, a marker of the cis-Golgi,43 the as Rab6A′ since four out of the five motifs involved organization of the Golgi apparatus was not in GTP binding are changed in Rab6C. Moreover, a significantly different in Rab6C-depleted cells, Rab6A′ mutant harboring a single amino acid which grow, however, in a disorganized manner substitution G25A in the P-loop motif was drasti- compared with control-treated cells (Fig. 8c). Quan- cally reduced in GTP binding ability. Before tification showed that the frequency of cells with concluding that the G-domain of Rab6C is nonfunc- multiple centrosomes was 24% in siRNA1-treated tional, however, further experiments are required cells compared to 2% in control cells at 96 h post- since Rab6C may operate as an unconventional transfection (Fig. 8d). Finally, to ensure that excess GTP-binding protein like other established excep- centrosomes are a phenotype caused by Rab6C loss, tions to the binary GTPase switch paradigm.44,45 we analyzed the number of centrosomes in HeLa Use of RNAi to study RAB6C function has also cells that do not express Rab6C, after treatment with been reported before.33 However, the short hairpin siRNA1 or control siRNA. Since siRNA1 also causes RNA targeted a sequence in RAB6C (termed WTH3 Rab6A′ depletion in HeLa cells, round cells in their study) that differs by only one nucleotide corresponding to cells blocked in metaphase and from RAB6A′, meaning that RAB6A′ was simulta- dead cells accumulate as a result of activated spindle neously knocked down in those studies. The assembly checkpoint proteins.27 Almost all siRNA1- observed increased tolerance of knockdown cells treated HeLa cells, however, contained one or two to doxorubicin could therefore be due to reduced centrosomal dots, similar to control-siRNA-treated levels of Rab6A′, previously implicated by the same cells (Fig. 8d). Hence, multiple centrosomes are not group to be involved in multidrug resistance.46 observed in a cell line that does not express Rab6C, Confusingly, Rab6A′ is termed as Rab6c in that supporting the view that supernumerary centro- study, which also describes original isolation of the somes are a specific effect caused by the depletion DNA methylated sequence corresponding to a of Rab6C. fragment of RAB6C (termed W3 in their study). The individual contributions of Rab6C and Rab6A′ Discussion to the phenomenon of multidrug resistance thus await future clarification.

Our results in the context of previous studies Rab6C is a rare example of a primate-specific concerning Rab6C retrogene demonstrated to express endogenous protein Our characterization of Rab6C highlights a num- ber of important differences to previous published The human genome encodes approximately the reports, most of which can be attributed to the fact same number of pseudogenes as genes but only a – that RAB6C is a retrogene. Notably, we show that small proportion are transcriptionally active.47 50 Of RAB6C has a tissue-restricted expression profile. these, only a few possess an intact ORF (18% in Ref. Presumably, part of the RAB6C signal detected in 49), indicating that the majority function as non- prior studies31,32,34 included contamination from protein-coding RNAs.51 The number of protein- genomic DNA, the authors being unaware that coding retrotransposed genes (referred to here as RAB6C transcript is colinear with its intronless retrogenes) contributing to the human proteome is retrogene. We could find no evidence of RAB6C unknown, largely due to the hypothetical status of expression in HeLa cells (Fig. 2a and b), which puts their protein-coding ability and absence of function- in question certain experiments regarding the al studies. While proteins encoded by older, regulation of the endogenous RAB6C promoter conserved retrogenes were often not initially recog- performed in this cell line.33 nized as belonging to this novel category of In previous studies,34 Rab6C and Rab6A/A′ intronless genes,52–54 in vivo experimental evidence levels were detected by immunoblotting cell lysates of endogenous protein encoded by more recently with an anti-Rab6A antibody from Santa Cruz emerged retrogenes is scarce. It is an important Biotechnology Inc. (Santa Cruz, CA). Surprisingly issue, however, as some expressed retrogenes to us, they found that Rab6C (termed WTH3 in their appear subject to posttranscriptional repression or study) is present at higher levels than Rab6A/A′ RNA surveillance55,56 and much skepticism exists (termed Rab6/6c in their study). There is no concerning the functional utility of recent retro- evidence showing that the Santa Cruz antibody genes. Since most retrogenes are probably low- detects bona fide Rab6C, whereas RNAi experiments abundance, differentially expressed, or develop- in our study unequivocally confirm the identity of mentally regulated proteins and in addition must the protein band we attribute to Rab6C. We be distinguished from their highly homologous therefore conclude that Rab6C is a low abundance counterparts, demonstration of endogenous expres- protein compared to Rab6A/A′. sion is a technically challenging task. RAB6C is one Rab6C was reported to be equally capable of of the first examples of a primate-specific retrogene binding GTP as Rab6A′,31 while our study con- for which the existence and functional relevance of cludes that Rab6C is an inefficient GTP-binding the endogenous protein has been addressed. A Retrogene with Functional Status 83

Rab6C at the centrosome The RAB6A′/RAB6C gene–retrogene pair gives rise to transcripts that are almost identical; It is interesting to consider how Rab6C, derived however, we managed to specifically study from a sequence originally encoding a Golgi- RAB6C function. Depletion of Rab6C resulted in associated Rab6A′ protein, underwent a potential tetraploidization and centrosome amplification, subcellular localization shift to the centrosome. We while there are no reports of excess centrosomes showed that the Rab domain of human Rab6C upon depletion of Rab6A′ or association of Rab6A′ – (GFP-Rab6C1 208) has diverged to the extent of at the centrosome. This suggests that RAB6C has having totally lost its ancestral ability to localize at evolved a new function derived from a RAB6A′ the Golgi. The 15-amino-acid substitutions scat- ancestral sequence by neo or subfunctionalization. tered throughout the atypical G-domain responsi- The resulting phenotype suggests that Rab6C is ble for centrosomal targeting of Rab6C (amino necessary for the successful completion of mitosis acids 1–174) presumably contributed to the subcel- or in the normal control of centrosome duplication. lular shift. We can also speculate that the C- In the literature, the most likely and frequent cause terminal extension made an important contribution of abnormal centrosome numbers is failed mitosis to the evolution of Rab6C. By inhibiting geranyla- due to defects in chromosome segregation or tion by the Rab escort protein/Rab geranylgeranyl improper cytokinesis.61,62 This results in a cell transferase, the C-terminal extension would have with 4N DNA content and two centrosomes, prevented ancestral Rab6C associating with the which, in the next S phase, will give a cell with Golgi and interacting with regulatory proteins such 8N DNA content and four centrosomes. Excess as guanine dissociation inhibitor (GDI) in the centrosomes can also arise via other mechanisms cytosol, which requires a prenylated Rab protein not associated with perturbed cell cycle function in the GDP-bound state.57 The C-terminal extension such as fragmentation of the pericentriolar may thus have released ancestral Rab6C from Golgi material,63 abnormal splitting of centrioles,64 and association/dissociation and the GDP/GTP cycle over-duplication of centrosomes.65 A cell with N3 and, no longer limited by its ancestral biological spindle poles that results from these processes will function, allowed Rab6C to acquire a new subcel- also consequently fail mitosis, giving rise to cells lular localization and function. A previous example with tetraploid 4N DNA content and multiple of subcellular relocalization was reported for an centrosomes. Further experiments are required to ape-specific retrogene CDC14Bretro.58 Through the distinguish if loss of Rab6C interferes with suc- use of GFP fusions and resurrection of ancestral cessful completion of mitosis/cytokinesis or cen- protein variants, the authors showed that substitu- trosome homeostasis. Amplification of centrosomes tions occurring throughout Cdc14retro lead to a is frequently observed in tumors and is thought to subcellular shift from microtubules to the cytosolic contribute to carcinogenesis by provoking multi- face of the endoplasmic reticulum. Adaptation of a polar mitoses, chromosomal instability, and protein to another subcellular compartment aneuploidy.66 Our results suggest that Rab6C requires changes in protein structure and probably indeed possesses tumor suppressor qualities and protein interacting partners. A recent two-hybrid offers an intriguing link to the reported frequent screen for novel Rab effectors that included Rab6C downregulation of Rab6C in multidrug-resistant failed to give any positive clones,59 and we cells.32 In conclusion, this study provides a encountered the problem that Rab6C is hardly convincing example of a retrotransposition event expressed in yeast transformants. Thus, a different that has generated a new gene and a novel technology will have to be used to identify Rab6C centrosomal-associated hominoid protein that protein partners. affects cell cycle progression. It should encourage the study of other recently emerged retrogenes that What is the role of RAB6C? exist in the human genome and were perhaps important to primate evolution or provide lineage- This article is the first to report a function of specific functions. Rab6C in processes related to cell cycle progression and regulation of centrosome number. Overexpres- Materials and Methods sion of Rab6C arrested cells in G1, preventing S phase entry of cells. Proteins that alter the compo- sition of the centrosome or pericentriolar material Antibodies and chemicals often cause G1-S arrest,60 and overexpression of Rab6C may inhibit the localization, signaling, or The following were used in this study: mouse anti-β enzymatic activity of other centrosomal proteins, tubulin clone TUB2.1 (1:5000; Sigma), rabbit anti-calnexin resulting in centrosome defects and a G1 block. (1:5000; Stressgen), mouse anti-GFP clones 7.1/13.1 Alternatively, the G1 arrest may be unrelated to any (1:1000; Roche), rabbit anti-GFP (1:1000; Invitrogen), rat anti-HA clone 3F10 (1:1000; Roche), mouse anti-HA.11 putative function of Rab6C at the centrosome; it may clone 16B12 (1:200; Convance), mouse anti-polyhistidine be related instead to a direct or indirect response clone HIS-1 (1:1000; Sigma), rabbit anti-GST (1:1000; of mammalian cells to overexpression that results in Invitrogen), mouse anti-Ki67 (1:1000; BD Transduction), an inhibition of cell cycle proteins involved in G1 mouse anti-GM130 (1:1000; BD Transduction), rabbit anti- progression. Rab6A/A′ (1:500; Santa Cruz), mouse anti-gamma tubulin 84 A Retrogene with Functional Status clone GTU88 (1:2000, Sigma), rat anti-BrdU (1:200; Identification of primate RAB6C orthologues from Immunologicals), rabbit anti-ninein,67 anti-AKAP450,68 genome databases rabbit anti-GDIβ,69 and rabbit anti-GMAP210.43 Second- ary antibodies included donkey anti-rabbit/mouse/rat RAB6C-related sequences (Supplementary Table S1) IgG conjugated to Cy3/Cy5 or horseradish peroxidase were obtained from the National Center for Biotechnology (HRP) (Jackson ImmunoResearch). DNA oligos were Information (NCBI) GenBank database with MEGABlast72 synthesized by Sigma-Genosys, siRNAs were synthesized or via the University of California Santa Cruz genome by Qiagen, and all sequences are listed in Supplementary browser with BLAT73 using intronless human RAB6C as Table S3. query. Since RAB6C is monoexonic, orthologues can be easily distinguished from the exon/intron containing ′ Cell culture, transfections, establishment of stable RAB6A/A and RAB6B genes. Human (H. sapiens) and cell lines, and immunofluorescence chimpanzee (Pan troglodytes) RAB6C sequences were downloaded from hg18 and panTro2. Whole genome shotgun and trace sequences of gorilla (Gorilla gorilla), HeLa, COS-7, Vero, RPE-1, and MCF-7 were grown in orangutan (Pongo abelii), and gibbon (Hylobates lar) were Dulbecco's modified Eagle's medium. LNCaP, SKBR-3, T- retrieved from NCBI Trace Archive†. Analysis of rhesus 47D, and EVSA-T were grown in RPMI 1640, and HEK293, macaque (Macaca mulatta), marmoset (Callithrix jacchus), HEK293T, and DU-145 were cultured in minimum and baboon (Papio anubis) genomes failed to return essential medium. Medium and supplements were pur- any RAB6C orthologues. Whole genome shotgun reads chased from Invitrogen. Medium was supplemented with μ were downloaded in fasta format, clipped, and aligned 2 mM glutamine, 150 g/ml penicillin, 100 U/ml using ClustalW.74 The multiple alignments were used to streptomycin, 1 mM sodium pyruvate, and 10% fetal calf deduce the putative amino acid sequence of each RAB6C serum (Pan Biotech). Cells were kept in an incubator at orthologue. 37 °C with 5% CO2. Transfection of plasmids was achieved using FuGENE 6 (Roche Diagnostics) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruc- RAB6C amplification from genomic DNA tions. Transfection of siRNA duplex oligonucleotides was performed using Lipofectamine RNAiMAX (Invitrogen). Chimpanzee genomic DNA was obtained from the Stable HEK293 cell lines expressing pIREShyg3GFPRab6C European Collection of Cell Cultures (ECACC repository, ′ or pIREShyg3GFP-siResRab6A were established by trans- Wiltshire, UK), and rhesus macaque genomic DNA was μ fection, selection, and amplification in 150 g/ml hygro- obtained from BioChain Institute (Hayward, CA, USA). mycin (Invitrogen). For immunofluorescence, HeLa were Genomic DNA from Chlorocebus aethiops (green monkey) grown on fibronectin/collagen or HEK293 were grown on was isolated from Vero and COS-7 cell lines using DNeasy poly(allylaminehydrochloride)-coated coverslips (Sigma) Blood and Tissue Kit (Qiagen). Primate DNA (150 ng) was – to 40% confluence. Where applicable, at 16 20 h after amplified with Taq polymerase (Invitrogen) and designed transfection, cells were fixed in 4% paraformaldehyde in primers. Resulting amplicons was cloned using the phosphate-buffered saline (PBS) (pH 7.2) for 15 min or pGEM-T Easy Vector System I (Promega) and plasmid − 20 °C cold methanol for 4 min. In prepermeabilization DNA purified from a number of recombinant clones using experiments, cells were extracted for 90 s in PHEM buffer Nucleospin (Macherey-Nagel). Bidirectional sequencing β [45 mM Pipes, 45 mM Hepes, 10 mM ethylene glycol bis( - was performed using the BigDye Terminator v3.1 Cycle ′ aminoethyl ether)N,N -tetraacetic acid, 5 mM MgCl2 Sequencing kit (ABI) in an ABI PROSM 3730XL DNA adjusted to pH 6.9, and 1 mM PMSF] containing 0.5% Analyzer system at the Curie Institute sequencing facility. Triton X-100 before fixation. To depolymerize microtu- bules, we incubated HeLa or RPE-1 cells expressing GFP- Rab6C in 33 μM nocodazole for 2 h at 37 °C and then on ice Directional RT-PCR for 30 min. For immunostaining, cells were incubated with primary antibody diluted in bovine serum albumin (0.2%) was examined in 20 human tissues and saponin (0.05%) in PBS for 1 h followed by washes in from the Human Total RNA Master Panel II (BD Clontech) PBS and another 1 h incubation with secondary antibody. or total RNAs extracted from cell lines using GenElute Cells were washed again in PBS and finally mounted on Mammalian Total RNA Miniprep columns (Sigma). RNA slides in Moviol. samples were systematically treated with Turbo DNA-free (Ambion) for 1 h at 37 °C to eliminate genomic DNA. PCR Plasmid constructions and mutagenesis was performed on these RNA samples using genomic specific primers (intronic PRSS-1 forward and reverse) to confirm absence of genomic contamination. RNAs were Control experiments used cells transfected with mito- reverse transcribed from 150 ng total RNA template, using GFP70 or H2B-GFP commercially available from BD AccessQuick RT-PCR system (Promega) and the appro- Biosciences. The ORF of RAB6C was PCR amplified priate reverse oligonucleotide at 45 °C for 45 min. AMV from clone DKFZp566K144 using high-fidelity Pfu DNA reverse transcriptase was inactivated at 95 °C for 2 min, polymerase (Promega) and directionally cloned using the forward oligonucleotide was applied, and single- standard techniques to create N-terminal GFP-, Cherry-, stranded cDNAs were amplified by PCR and 25–30 GST-, MBP-, and His-tagged fusions. HA-Rab6C was repeated cycles of 95 °C for 30 s, 62 °C for 30 s, and created by cloning RAB6C into a modified pcDNA vector 72 °C for 45 s followed by a final elongation step at 72 °C containing a 4xHA epitope tag (gift from Ole Vielemeyer, for 5 min. Products were analyzed on 1% agarose gels. To DUCOM, Philadelphia). To create GFP-Rab6A+ext chi- test the specificity of the primers, controls consisted of mera, we used a two-step PCR strategy, while GFP- templates encoding the complete cDNA including UTR Rab11A+ext was constructed by PCR-mediated ligation.71 Site-directed mutagenesis was performed according to the QuikChange protocol (Stratagene). All constructs were verified by sequencing. † http://www.ncbi.nlm.nih.gov/Traces/trace.cgi? A Retrogene with Functional Status 85 regions obtained from the following sources: RAB6C mm glass dishes (Iwaki). At 24 h post-transfection, cells AL136727/DKFZp566K144 from DKFZ (Heidelberg, Ger- were imaged in a CO2-controlled live-cell chamber ′ many), RAB6A BC003617/MGC:1654 IMAGE:3506585 equilibrated at 5% CO2 and 37 °C (Life Imaging Services). from RZPD (Berlin, Germany), RAB6B BX442004/ Time-lapse sequences were recorded every 15-min for 26 h CS0DF024YC24 from Genoscope (Evry, France), and using an inverted Nikon TE2000-E microscope with 40× RAB6A pUC8RAB6A.75 0.6 NA objective. All microscopes were equipped with a cooled charge-coupled device camera (Coolsnap HQ, Photometrics) and controlled by Metamorph software Cell synchronization, flow cytometry analysis, and (Universal Imaging). BrdU incorporation

Cells were arrested in S phase by treatment with 2.5 mM Photobleaching analysis thymidine for 16 h twice with a 9-h interval of growth without the drug or synchronized in G2 by releasing from HeLa Centrin1-GFP cells transfected 16–20 h earlier S arrest for 9 h. For mitotic arrest, the cells were first with a plasmid encoding Cherry-Rab6C were put just treated with 2.5 mM thymidine for 16 h and then treated prior to the experiment in non-phenol-red-containing with 5 μM nocodazole (Sigma) for 10 h. Cells were Dulbecco's modified Eagle's medium supplemented resuspended in 70% ethanol, incubated with propidium with 10% calf serum plus 25 mM Hepes (Invitrogen). iodide (Sigma) and RNase, and analyzed by FACSCalibur Coverslips were placed in a heated chamber at 37 °C for (BD Biosciences). To quantify DNA content histograms, the duration of the experiments. Centrosomes were we analyzed FACS data by ModFit software (Verity Inc.). imaged with a 60× 1.4 NA objective using a Nikon C1si BrdU was added at 40 μM for 10 min to cells kept in confocal laser scanning microscope based on an inverted culture medium before fixing in PAF or MeOH to quantify TE2000 microscope. Photobleaching experiments of GFP the number of transfected cells in S phase. Cells were fusions were done using the 488-nm argon laser and denatured with 2 N HCl at room temperature for 10 min Cherry-Rab6C using the 561-nm line of this system at and washed thrice with PBS, prior to immunostaining of 100% intensity and controlled with the Ez-C1 Nikon BrdU-incorporated DNA. software. Briefly, a 30-μm region containing the centro- some was exposed to three consecutive pulses such that 70–80% of the fluorescent protein signal was lost after Protein stability, prenylation assays, GST-Rab bleaching. Five images were captured prior to bleaching purification, and GTP binding assays and in 10-s increments for 250 s or every second for 70 s postbleach. Intensity data values of the photobleached μ To determine the half-life of Rab6C, we added 100 g/ centrosome were exported into Microsoft Excel and ml cycloheximide (Sigma) to HEK293 cell cultures to corrected for overall photobleaching by dividing by the inhibit protein synthesis, and cells were harvested at the average intensity of a defined area within the same cell indicated time intervals. HEK293 cells can be lifted rapidly that was of a reasonable distance from the photobleached from culture dishes by simply pipetting medium over centrosome. them. To determine the effect of inhibitors on Rab6C stability, cells were preincubated with lysosomal inhibi- μ μ Structural model of Rab6C, antibody generation, tors chloroquine (100 M; Sigma) or leupeptin (50 M; and detection of Rab6C Euromedex) or proteasome inhibitors MG-132 (10 μM) or MG-115 (3 mM) purchased from Calbiochem for 9 h before harvesting. To evaluate the ability of GFP-Rab6C and The sequence of the unconventional G-domain of human Rab6C (residues F14–P175) was overlaid onto derivatives to undergo geranylation, we subjected trans- ′ fected HeLa cells to Triton X-114 phase-partitioning assays the crystal structure of the GTP-bound form of Rab6A at 76 1.78 Å resolution (Protein Data Bank ID: 1YZQ; residues as described previously. GST fusion proteins were – expressed in BL21 Rosetta (Novagen) and purified on F14 P175). Sixteen positions (Ala25, Arg34, Ile45, Gly60, glutathione Sepharose 4B beads (GE Healthcare) as Gly63, Arg65, Leu75, Arg81, Asp94, Arg115, Thr122, described previously.77 Binding of [α-32P]GTP (Perkin Arg127, Val138, Gly147, Thr151, and Arg156) were mutated, and stereochemistry regularization of the Elmer) to blotted GST-Rab fusion proteins was performed 79 as described previously.78 overall model was performed using Coot. The anti- Rab6C antibody was generated by immunizing rabbits (AGRO-BIO) with His-Rab6C purified from inclusion Fixed and live-cell microscopy bodies in the presence of 8 M urea on Ni-NTA agarose beads according to the manufacturer's protocols (Qia- Immunofluorescence images were acquired with an gen), followed by concentration and stepwise dialysis to epifluorescent microscope (DMRA, Leica Microsystems) 4 M urea in PBS+50% glycerol. Serums were affinity- using 40× 1.25 NA and 63× 1.32 NA objectives. Decon- purified on MBP-Rab6C antigen-coupled Sepharose volved images were obtained with a wide-field micro- column. The MBP-Rab6C was expressed as a soluble scope (DRMA, Leica) using a 100× 1.4 NA Plan Apo protein and purified according to standard procedures objective coupled to a piezo motorized stage, enabling on amylose resin (New England Biolabs). To immuno- acquisition of images every 200 nm in the Z plane. Stacks precipitate Rab6C, we extracted cells in ice-cold lysis of nonsaturated images (15–20 planes) were deconvolved buffer (10 mM Tris, pH 8, 1 mM ethylenediaminetetraa- using Meinel algorithm with Metamorph software (Uni- cetic acid, 0.5% NP-40, 0.5% DOC, and 1 mM PMSF) versal Imaging). Maximum or average intensity flattened including protease inhibitor cocktail (Sigma) and briefly images were then extracted with ImageJ‡ to obtain the sonicated. One milligram of cleared lysates was sub- final views. For live imaging, cells were transfected in 35- jected to immunoprecipitation with rabbit anti-Rab6C polyclonal antibody at 4 °C overnight. Protein A- Sepharose CL-4B beads (GE Healthcare) were added ‡ http://rsb.info.nih.gov/ij/ for 2 h, and immunocomplexes were washed 3 times in 86 A Retrogene with Functional Status

0.1% NP-40 in PBS and eluted by boiling in SDS sample 5. Grigoriev, I., Splinter, D., Keijzer, N., Wulf, P. S., buffer. Proteins were resolved on 12% SDS-PAGE gels Demmers, J., Ohtsuka, T. et al. (2007). Rab6 regulates and transferred electrophorectically to nitrocellulose transport and targeting of exocytotic carriers. Dev. filters and immunoblotted with anti-Rab6C and Cell, 13, 305–314. proteinAHRP (1:8000; Zymed-CliniSciences), which binds 6. Racine, V., Sachse, M., Salamero, J., Fraisier, V., preferentially to the Fc portion of intact IgGs. Bound Trubuil, A. & Sibarita, J. B. (2007). Visualization and antibodies were detected using SuperSignal West Femto quantification of vesicle trafficking on a three-dimen- enhanced chemiluminescence kit (Thermo). sional cytoskeleton network in living cells. J. Microsc. 225, 214–228. 7. Luo, Z. & Gallwitz, D. (2003). Biochemical and genetic evidence for the involvement of yeast Ypt6-GTPase in protein retrieval to different Golgi compartments. 278 – Acknowledgements J. Biol. Chem. , 791 799. 8. Miserey-Lenkei, S., Waharte, F., Boulet, A., Cuif, M. H., Tenza, D., El Marjou, A. et al. (2007). Rab6-interacting We thank S. Wiemann (DKFZ, Heidelberg, Ger- protein 1 links Rab6 and Rab11 function. Traffic, 8, many) for RAB6C cDNA and R. Pepperkok (Euro- 1385–1403. pean Molecular Biology Laboratory, Heidelberg, 9. Monier, S., Jollivet, F., Janoueix-Lerosey, I., Johannes, Germany) in whose laboratory this work was L. & Goud, B. (2002). Characterization of novel Rab6- initiated, A Girod (European Molecular Biology interacting proteins involved in endosome-to-TGN Laboratory) for help with cloning, G. Langsley transport. Traffic, 3, 289–297. (Institute Cochin, Paris) for pEGFPRab11A, M. 10. Mallard, F., Tang, B. L., Galli, T., Tenza, D., Saint-Pol, Bornens (Institute Curie, Paris) for HeLa Centrin1- A., Yue, X. et al. (2002). Early/recycling endosomes-to- GFP cell line, F. Radvanyi (Institute Curie) for TGN transport involves two SNARE complexes and a Rab6 isoform. J. Cell Biol. 156, 653–664. normal human breast RNA, N. LeKieffre for 11. Liewen, H., Meinhold-Heerlein, I., Oliveira, V., purification of His-Rab6C, A. El Marjou and L. Schwarzenbacher, R., Luo, G., Wadle, A. et al. (2005). Cabanie for anti-Rab6C antibody purification, Z. Characterization of the human GARP (Golgi associated Maciorowski for expert FACS analysis, Institute retrograde protein) complex. Exp. Cell Res. 306,24–34. Curie Cell and Tissue Imaging Facility for help with 12. Del Nery, E., Miserey-Lenkei, S., Falguieres, T., Nizak, microscopy, and R. Basto for critical reading of the C., Johannes, L., Perez, F. & Goud, B. (2006). Rab6A manuscript. 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