A Family of New Mammalian Orthologues of Yeast Sec31p That Associate with the COPII Coat

958 Research Article Human Sec31B: a family of new mammalian orthologues of yeast Sec31p that associate with the COPII coat

Michael C. Stankewich*, Paul R. Stabach* and Jon S. Morrow‡ Departments of Pathology and Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 19 October 2005 Journal of Cell Science 119, 958-969 Published by The Company of Biologists 2006 doi:10.1242/jcs.02751

Summary We have cloned human brain and testis Sec31B protein presumably binds Sec23/24. The Sec31B-T group (also known as secretory pathway component Sec31B-1 or (molecular mass 52,983 Da) contains a preserved WD- SEC31-like 2; GenBank accession number AF274863). repeat domain but lacks the C-terminal proline-rich region. Sec31B is an orthologue of Saccharomyces cerevisiae When expressed as a fusion protein with eYFP in cultured Sec31p, a component of the COPII vesicle coat that cells, Sec31B-F associates with the endoplasmic reticulum mediates vesicular traffic from the endoplasmic reticulum. and with vesicular-tubular clusters, displays restricted Sec31B is widely expressed and enriched in cerebellum and intracellular movement characteristic of COPII vesicle testis. Its predicted sequence of 1180 residues (expected dynamics, co-distributes on organelles with Sec13, Sec31A molecular mass 128,711 Da) shares 47.3% and 18.8% and Sec23 (markers of the COPII coat), and concentrates similarity to human Sec31A (also known as Sec31; with ts045-VSV-G-CFP (VSV-G) when examined early in GenBank accession number AF139184) and yeast Sec31p, the secretory pathway or after temperature or nocodazole respectively. The gene encoding Sec31B is located on inhibition. The role of the truncated form Sec31B-T chromosome 10q24 and contains 29 exons. PCR analysis of appears to be distinct from that of Sec31B-F and remains exon utilization reveals massive alternative mRNA splicing unknown. We conclude that Sec31B-F contributes to the of Sec31B, with just 16 exons being constitutively utilized diversity of the mammalian COPII coat, and speculate that in all transcripts. The presence of a stop codon in exon 13 the Sec31 cage, like Sec24, might be built with isoforms generates two families of Sec31B gene products (each tuned to speciﬁc types of cargo or to other specialized

Journal of Cell Science displaying additional patterns of mRNA splicing): a group functions. of full-length proteins (hereafter referred to as Sec31B-F) and also a group of truncated proteins (hereafter referred Supplementary material available online at to as Sec31B-T), distinguished by their utilization of exon http://jcs.biologists.org/cgi/content/full/119/5/958/DC1 13. Sec31B-F closely resembles Sec31p and Sec31A, with canonical WD repeats in an N-terminal domain that binds Key words: ER, Secretory pathway, COPII, Vesicle, Golgi, Secretion, Sec13 and a proline-rich C-terminal region that Protein isoforms

Introduction recruitment of accessory proteins necessary for vesicle Entry to the secretory pathway is initiated by the extrusion of targeting and docking (e.g. SNAREs) (Matsuoka et al., 1998; COPII-coated cargo-loaded transport vesicles and tubules Springer and Schekman, 1998; Mossessova et al., 2003); and from the endoplasmic reticulum (ER). The basic outlines of (3) the Sec13-Sec31 protein complex, which is recruited to COPII coat formation and the extrusion process are now complete the COPII coat and has an effect on vesicle budding understood in considerable detail (for reviews see Springer et (Salama et al., 1997). Sec13-Sec31 is a putative bivalent al., 1999; Brittle and Water, 2000; Klumperman, 2000; heterotetramer believed to cross-link pairs of Sar1p-Sec13- Antonny and Schekman, 2001; Bickford et al., 2004). As first Sec31 complexes into a 2D vesicle coat (Lederkremer et al., established in S. cerevisiae by genetic analysis, and later 2001; Matsuoka et al., 2001). confirmed by biochemical and ultrastructural studies in both Whereas the basic features of COPII coat formation (as yeast and in other species, cargo sorting and vesicle budding described above) are evolutionarily well conserved, from the ER is driven by three cytosolic complexes containing complexities in the secretory pathway are increasingly being a total of five proteins: (1) the small GTP-binding protein recognized in higher organisms. In mammals, the packaging of Sar1p initiates coat formation at specialized ER exit sites procollagen, a molecule too large to fit into a 60-85 nM COPII (ERES) (Bannykh et al., 1996; Aridor et al., 2001); (2) the vesicle, appears to exit the ER through en bloc protrusion of a heterodimeric complex of Sec23-Sec24 binds to Sar1-GTP patch of ER membrane that is devoid of a COPII coat (although and provides for cargo selection (Aridor et al., 1998; Miller et this process only occurs adjacent to COPII-coated ERESs and al., 2003), membrane bending (Bi et al., 2002), and the depends on Sar1) (Mironov et al., 2003). Chylomicrons may Sec31B alternative transcripts 959

also exit the ER in a similar manner (Siddiqi et al., 2003; Results Shoulders et al., 2004). GPI-linked proteins exit by yet a third Identification of human Sec31B and the exon structure unique pathway that involves their sorting into sphigolipid-rich of Sec31B rafts in the ER (Muniz et al., 2001). The cystic fibrosis The gene for Sec31B was first identified in silico by pair-wise transmembrane conductance regulator (CFTR) exits the ER by comparison of rat Sec31 aa sequences (Shugrue et al., 1999) a COPII-dependent pathway and transits to the Golgi network with the high-throughput genomic-sequence-database using in a non-conventional way, possibly trough an endosomal TBLASTN (http://www.ncbi.nlm.nih.gov/BLAST). A match compartment (Yoo et al., 2002). Spectrin and ankyrin, was made with GenBank Homo sapiens chromosome 10, clone cytoskeletal proteins that are absent in yeast, play a role in RP11-411B6 (GenBank accession number AL133352) mammalian cells in determining the efficiency of transport of deposited by the Sanger Centre. In addition, 38 sequence selected proteins through the secretory pathway, including even matches were identified from the dbESTs, of which five were the efficiency of their exit from the ER and their pathway of from both testis and ovary libraries. One EST matched a partial intracellular targeting and/or retention (Devarajan et al., 1997; clone (accession number AL080141) in the non-redundant Tuvia et al., 1999; De Matteis and Morrow, 2000; Pradhan and nucleotide database submitted by the German Cancer Research Morrow, 2002). Such findings suggest that evolution has Center. Exon mapping of adjacent genomic sequences allowed modified and added to the basic ER-exit and -sorting formula identification of WNT8B as an adjacent gene; this allowed found in yeast, to meet the more complex and specialized needs mapping of Sec31B to locus 10q24. Oligonucleotides were of higher organisms. One way evolution modifies function is designed to amplify contigs using Marathon-Ready cDNA to develop protein orthologues and isoforms, e.g. by gene libraries from human testis, brain and fetal brain. Sequence at duplication and/or by alternative mRNA splicing. Both the 5Ј-end was extended by RACE amplification. The mechanisms appear to have been used in refining the oligonucleotides used to span the sequence of Sec31B are mechanisms of ER export. summarized in supplementary material Fig. S1. All reaction Mammalian orthologues have been identified for each of the products from each library were bi-directionally sequenced five proteins involved in COPII coat formation, and in some (Keck Biotechnology Laboratories, Yale University) from at cases, multiple isoforms of these proteins exist. For example, least three independent amplifications to eliminate PCR- two mammalian isoforms of Sar1 and Sec23, and four derived errors. All libraries demonstrated an abundance of mammalian isoforms of Sec 24 have been reported (Kuge et alternative transcripts (see below). The full-length cDNA al., 1994; Paccaud et al., 1996; Pagano et al., 1999; Tang et al., transcript identified from this analysis and identified as 1999). Only one form of mammalian Sec13 has been described Sec31B, was deposited under the GenBank accession number (Shaywitz et al., 1995). Two groups have independently AF274863. This full-length cDNA comprises 4648 nucleotides identified the mammalian protein corresponding to the Sec31p (nts), and includes 5Ј- and 3Ј-untranslated regions of 98 and yeast protein, named Sec31A (Shugrue et al., 1999; Tang et al., 1010 nts, respectively. The open-coding-region consists of 2000). It has also been noticed, by examining published 3540 nts, predicting a 1180 amino acid (aa)-long protein with expressed sequence tags (ESTs), that a second mammalian the molecular mass of 128,711 Da (Fig. 1). A satisfactory

Journal of Cell Science orthologue of Sec31p, named Sec31B, exists that has not yet Kozak initiation sequence is present immediately upstream of been characterized (Tang et al., 2000). With the exception of a the first ATG and an in-frame stop codon occurs at nt 93. We putative role in mediating cargo specificity (Shimoni et al., are thus confident that the second ATG at nt 99 is the initiator 2000), and a single report of Sec23-Sec24 participation in methionine of the derived protein. We have termed the derived endocytosis (Penalver et al., 1999), no specific role for isoform protein from this transcript Sec31B-F, in recognition of its full- variation in the COPII composition has been identified or length status and the existence of shorter truncated peptides proposed. that arise by alternative exon usage from this gene (see below). In the present study we have cloned and characterized A comparison of the predicted aa sequence of Sec31B-F human Sec31B and determined the intron-exon structure of the with human Sec31A and with S. cerevisiae Sec31p reveals the gene encoding Sec31B (also known as Sec31L2). We present greatest degree of homology over the N-terminal half of the evidence that, in addition to being widely expressed in protein, the region that contains seven canonical WD repeats. mammalian tissues, mRNA transcripts of Sec31B (also known Its homology to Sec31p is reduced in the proline-rich regions as secretory pathway component Sec31B-1 or SEC31-like 2; distributed over its C-terminal half (sequences involved with GenBank accession number AF274863) display considerable the binding of Sec23). Overall, Sec31B-F shares 47.3% and alternative mRNA splicing, generating a range of highly 18.8% identity to human Sec31A and yeast Sec31p, divergent protein products that fall into two general categories, respectively. several alternative versions of full-length Sec31B proteins with Search of the genome database with the complete Sec31B a mass of about 129 kDa, and a group of truncated proteins sequence revealed a match with the Sanger Centre Homo including one of about 53 kDa (hereafter referred to as sapiens clone RP11-411B6 (GenBank accession number Sec31B-T). The full-length gene products are similar to AL133352) and allowed its genomic structure to be deduced Sec31A in structure, disposition and their apparent function, (Fig. 2). The gene encoding Sec31B is located at chromosomal and appear to be bona fide components of the mammalian locus 10p24, incorporates 29 exons, and spans 33,187 bp. As COPII coat. The 53 kDa gene product assumes an intracellular described below, the gene is subject to considerable alternative distribution distinct from Sec31A. Its role remains unknown exon utilization. Of the 29 exons, 16 were found in all and will be addressed in future studies. The focus of the present transcripts; these exons appear to be constitutively expressed study is on a full-length Sec31B protein (hereafter referred to and are depicted as green in Fig. 2. The other 13 exons as Sec31B-F). (represented in red) are quite variably expressed. Exon 19 has 960 Journal of Cell Science 119 (5)

Sec31p yeast MVKLAEFSRTATFAWS-HDKIPL-LVSGTVSGTVDANFSTDSSLELWSLLAADSE---KPIASLQVDSKFNDLDWS----HNNK---IIAGAL----DNG 84 Sec31A Human M-KLKEVDRTAMQAWSPAQNHPIYLATGTSAQQLDATFSTNASLEIFELDLSDPSLDMKSCATFSSSHRYHKLIWGPYKMDSKG---DVSGVLIAGGENG 96 Sec31B Human M-KLKELERPAVQAWSPASQYPLYLATGTSAQQLDSSFSTNGTLEIFEVDFRDPSLDLKHRGVLSALSRFHKLVWG----SFGSGLLESSGVIVGGGDNG 95 Sec31B-t Sec31p yeast SLELYSTNE--ANNAINSMARFSNHSSSVKTVKFNAKQDNVLASGGNNGEIFIWDMNKCTESPSNYTPLTPGQSMS-SVDEVISLAWNQSLAHVFASAGS 181 Sec31A Human NIILYDPSKIIAGDKEVVIAQNDKHTGPVRALDVNIFQTNLVASGANESEIYIWDLNNFA------TPMTPGAKTQ-PPEDISCIAWNRQVQHILASASP 189 Sec31B Human MLILYNVTHILSSGKEPVIAQKQKHTGAVRALDLNPFQGNLLASGASDSEIFIWDLNNLN------VPMTLGSKSQQPPEDIKALSWNRQAQHILSSAHP 189 Sec31B-t Sec31p yeast SNFASIWDLKAKKEVIHLSYTSPNSGIKQQLSVVEWHPKNSTRVATATGSDNDPSILIWDLRNANTPLQTLNQGHQKGILSLDWCHQDEHLLLSSGRDNT 281 Sec31A Human SGRATVWDLRKNEPIIKVS----DHSNRMHCSGLAWHPDVATQMVLASEDDRLPVIQMWDLRFASSPLRVLEN-HARGILAIAWSMADPELLLSCGKDAK 284 Sec31B Human SGKAVVWDLRKNEPIIKVS----DHSNRMHCSGLAWHPDIATQLVLCSEDDRLPVIQLWDLRFASSPLKVLES-HSRGILSVSWSQADAELLLTSAKDSQ 284 Sec31B-t Sec31p yeast VLLWNPESAEQLSQFPARGNWCFKTKFAPEAPDLFACASFDNKIEV------QTLQNLTNTLDEQET-ETKQQESETDFWNNVSREESKEKPS 367 Sec31A Human ILCSNPNTGEVLYELPTNTQWCFDIQWCPRNPAVLSAASFDGRISVYSIMGGSTDGLRQKQVDKLSSSFGNLDPFGTGQPLPPLQ-----IPQQTAQHSI 379 Sec31B Human ILCRNLGSSEVVYKLPTQSSWCFDVQWCPRDPSVFSAASFNGWISLYSVMGRSWEVQHMRQADKISSSFSK------GQPLPPLQ-----VPEQVAQAPL 373 Sec31B-t Sec31p yeast VFHL-QAPTWYGEPSPAAHWAFGGKLVQIT----PDGKGVSITNPK------ISG------LESNTTLSEALKTKDFKPLINQRLVKVIDDVNEE 445 Sec31A Human VLPLKKPPKWIRRPVGASF-SFGGKLVTFENVRMPSHQGAEQQQQQ------HHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKIDASQTEFEKN 472 Sec31B Human IPPLKKPPKWIRRPTGVSF-AFGGKLVTFG----LPSTPAHLVPQPCPRLVF----ISQVTTESEFLMRSAELQEALGSGNLLNYCQNKSQQALLQSEKM 464 Sec31B-t Sec31p yeast DWNLLE-KLSMDGTEEFL------KEAL------AFD------NDESDA---QDDANNEKED-DGEEFF------QQIET 496 Sec31A Human VWSFLKVNFEDDSRGKYLELLGYRKEDLGKKIAL------ALNKVDGANVALKDSDQV---AQSDGEESPA-AEEQLLGEHIKEEKEES 551 Sec31B Human LWQFLKVTLEQDSRMKFLKLLGYSKDELQKKVATWLKSDVGLGESPQPKGNDLN------SDRQQAFCSQASKHTTKEASASSAFF------DELVP 549 Sec31B-t KPARDSSGARR Sec31p yeast NFQPEG----DFSLSGNIEQTISKNLVSGNIKSAVKNSLENDLLMEAMVIALDSNNERLKESVKNAYFAKYGSKSSLSRILYSISKREVDDLVENLDVSQ 592 Sec31A Human EFLPSSGGTFNISVSGDIDGLITQALLTGNFESAVDLCLHDNRMADAIILAIAGGQE-LLARTQKKYFAK--SQSKITRLITAVVMKNWKEIVESCDLKN 648 Sec31B Human QNMTPW----EIPITKDIDGLLSQALLLGELGPAVELCLKEERFADAIILAQAGGTD-LLKQTQERYLAK--KKTKISSLLACVVQKNWKDVVCTCSLKN 642

Sec31p yeast WK-FISKAIQNLYPNDIAQRNEMLIKLGDRLKENGHR--QDSLTL-YLAAGSLDKVASIWLSEFPDLEDKLKKDNKTIYEAHSECLTEFIERFTVFSNFI 688 Sec31A Human WREALAAVLTYAKPDEFSALCDLL---GTRLENEGDSLLQTQACLCYICAGNVEKLVACW------TKAQDGSHPLSLQDLIEKVVILRKAV 731 Sec31B Human WREALALLLTYSGTEKFPELCDML---GTRMEQEGSRALTSEARLCYVCSGSVERLVECW------AKCHQALSPMALQDLMEKVMVLNRSL 725

Sec31p yeast N---GSSTINNEQ----LIA-KFLEFINLTTSTGNFELATEFLNSLPSDNEEVKTEKARVLIASGKSLPAQNPATATTSKAKYTNAKTNKNVPVLPTPGM 780 Sec31A Human Q---LTQAMDTSTVGVLLAA-KMSQYANLLAAQGSIAAALAFLPD-NTNQPNIMQLRDRLCRAQGEPVAGHESPKIPYEKQQLPKGRPGP------V 817 Sec31B Human EQLRGPHGVSPGP----ATTYRVTQYANLLAAQGSLATAMSFLPR-DCAQPPVQQLRDRLFHAQGSAVLGQQSPPFPFPRIVVGATLHSK------E 811

Sec31p yeast PSTTSIPSMQAPFY---GMTPGASA----NALPPKPYVPATTTSAP------VHTEG-KY-APPS--QPSMASPFVNKTNSSTRLNSFAPPPNPYATA 861 Sec31A Human AGHHQMPRVQTQQYYPHGENPPPPGFIMHGNVNPNAAGQLPTSPGH------MHTQVPPY-PQPQPYQPAQPYPFGTGGSAMYRPQQPVAPPTSNAYP 908 Sec31B Human TSSYRLGSQPSHQ----VPTPSPRP----RVFTPQSSPAMPLAPSHPSPYQGPRTQNIS-DYRAPGP--QAIQPLPLSPGVRPASSQPQLLGGQRVQVPN 900

Sec31p yeast TVPATNV-STTSIPQNTFAPIQPGMPIMGDYNAQSSSIPSQPP-INAVSGQTPHLNRKANDGWNDLPLKVKEKPSRAKAVS-----VAPP--NILSTPTP 952 Sec31A Human NTPYISS-ASSYTGQSQLYAAQH------QASSPTSSPATS-FPPPPSSGASFQHGGPGA------PPSSSAYALP-----PGTT--GTLPAASE 982 Sec31B Human PVGFPGTWPLPGSPLPMACPGIM------RPGSTSLPETPRLFPLLPLRPLGPGRMVSHT------PAPPASFPVPYLPGDPGAPCSSVLPTTGI 983 Journal of Cell Science Sec31p yeast LNGIPANAAST--MPPPPLSRAPSSVSMVSPPPLHKNSRVPSLVATSESPRASISNPYAPPQSS--QQFPIGTISTANQTS-NTAQVASSNPYAPPPQQR 1047 Sec31A Human LPASQRTGPQNGWNDPPALNRVP------KKKKMPENFMPPVPITSPIMNPLGDPQSQMLQQQPSAPVPLSSQSSFPQPHLPGGQPF-----HG 1065 Sec31B Human L--TPHPGPQDSWKEAPAPRGNL------QRNKLPETFMPPAPITAPVMS-LTP------ELQGILPSQPPVS-SVSHAPPGVP------1052

Sec31p yeast VATPLSGGVPPAPLPKASNPYAPTATTQPNGSSYPPTGPYTNNHTMTSPPPVFNKPPTGPPPISMKKRSNKLASIEQNPSQ--GATYPPTLSSSASPLQP 1145 Sec31A Human VQQPLGQ------TGMPPSFSKP------NIEGAPGAPIGNTFQHVQSLPTKKITK 1109 Sec31B Human -----GE------LSL------QLQHLPPEKMER 1068

Sec31p yeast SQPPTLASQVNTSAENVSHE-IPADQQPIVDFLKEELARVTPLTPKEYSKQLKDCDKRLKILFYHLEKQDLLTQPTIDCLHDLVALMKEKKYKEAMVIHA 1244 Sec31A Human KPIPDEHLILKTTFEDLIQRCLSSATDPQT------KRKLDDASKRLEFLYDKLREQTLSPTITSG-LHNIARSIETRNYSEGLTMHT 1190 Sec31B Human KELPPEHQSLKSSFEALLQRCSLSATDLKT------KRKLEEAAQRLEYLYEKLCEGTLSPHVVAG-LHEVARCVDAGSFEQGLAVHA 1149

Sec31p yeast NIATNHA-QEGGNWLTGVKRLIGIAEA-TLN 1273 Sec31A Human HIVSTSNFSETSAFMPVLKVVLTQANKLGV. 1221 Sec31B Human QVAGCSSFSEVSSFMPILKAVLIIAHK-LLV 1179

Fig. 1. Sequence alignment of human Sec31B-F. The derived aa sequence of the human Sec31B-F (GenBank accession number AF274863) aligned with human Sec31A (Tang et al., 2000) and Sec31p (Salama et al., 1997) by the program MegAlignTM (DNAStar) using the J. Hein algorithm. Identical residues are shaded. Sec31B shares 47.3% and 18.8% identity to human Sec31A and yeast Sec31p respectively. The bar indicates the extent of Sec31B-T, the truncated isoform derived from utilization of exon 13. This isoform retains the WD-repeat region found in the N-terminal half of the protein, responsible for Sec13 binding, but lacks the C-terminal region responsible for Sec23-Sec24 binding (Shaywitz et al., 1997).

two putative splice-donor sites, verified by their presence in in Fig. 2C. This is the longest transcript observed identified transcripts, and is therefore represented as 19A experimentally. Also depicted are the exons translated to (closed) and 19B (open). Each intron-exon junction of human generate the two antibodies (Mab 1D4 and Mab 1G10) used in Sec31B is displayed in Fig. 2 with 20 base pairs of flanking this study, both directed against sequences derived from exons sequence. The pattern of exon utilization of the Sec31B-F 5 to 13, IgG 2013 directed against sequences in exons 16 and isoform as represented by GenBank (AF274863) is presented 17, and IgY 1871, directed to sequences derived from exons Sec31B alternative transcripts 961

16 through 29. Efforts to generate an antibody to the unique were submitted separately to GenBank: exon 12, AF279137; sequence in Sec31B-T (see below), based on the 11 C-terminal exon 13, AF279138; exon 19B, AF279139 (only detected residues of this isoform derived from exon 13, have so far been once, representing an alternative splice-donor at the 3Ј-end of unsuccessful. exon 19); exon 21, AF279140. The frequency of observation of each exon in the transcripts examined is presented Human Sec31B generates multiple transcripts by schematically in Fig. 3A. These frequencies also parallel alternative mRNA splicing closely the frequency with which such transcripts are observed Three cDNA libraries were exhaustively examined for alternate in EST data available in GenBank. An area of micro-diversity exon utilization by PCR (supplementary material Fig. S1). The was noted at the 3Ј splice junction of exon 5, in which one Sec31B-F transcript (AL133352) utilized 26 of the 29 exons codon would be deleted. This was detected in seven of 16 in the gene (Fig. 2C). Other exons not included in AL133352 independent amplimers. Examples of four alternatively spliced Journal of Cell Science

Fig. 2. Genomic structure of human Sec31B. The gene is located on chromosome 10p24, spans 33,187 bp and incorporates 29 exons. (A) Constitutively expressed exons are depicted as green boxes; alternatively utilized exons as red boxes. The size of each exon is drawn to scale. Arrows indicate the start-codon in exon 2 and the stop-codon in exon 29; these sites are utilized in Sec31B-F. Exon 19 has two putative splice-donor sites and is therefore represented as 19A (closed) and 19B (open). (B) Each intron-exon junction of human Sec31B is displayed with 20 base pairs of ﬂanking sequence. The ﬁrst exon begins with the adapter primer used for 5Ј RACE and the last exon ends with a polyA tail. Lower- and upper-case letters denote intron and exon sequences, respectively (colored as above). (C) Exon utilization of the Sec31B-F isoform as represented by GenBank (AF274863). This is the longest transcript observed experimentally, predicted to encode a 129 kDa protein. Also depicted are the exons that had been translated to generate the antibodies used in this study. Mab1D4 (shown) and Mab1G10 were both raised against the same exons. 962 Journal of Cell Science 119 (5)

transcripts depicting their exon utilization are shown (Fig. 3B), exon 4 is not utilized, then exon 5 contributes an out-of-frame together with a representation of their open reading frames. stop codon. Thus, much shorter Sec31B peptides might also Of signiﬁcant interest was the frequent utilization of exon exist (because exon 4 is not constitutively utilized but exon 5 13. This exon, detected in 23 of 27 transcripts that ﬂanked this is). However, we have no other direct evidence for such very region, contains an in-frame stop codon; its incorporation is short expression products. predicted to generate a truncated protein product with the Although it is not possible in the absence of full-length molecular mass of 52,983 Da. The predicted translation messages for every transcript to determine the precise products of Sec31B thus fall into two general categories. Those combinatorial patterns of exon utilization that actually occur, without exon 13 are expected to generate a family of it is clear that many variations exist. Fig. 3C presents a cartoon alternatively spliced ‘full-length’ proteins (e.g. Sec31B-F). summarizing the patterns of exon utilization that have been Conversely, transcripts incorporating exon 13 will generate experimentally observed to date, along with the approximate truncated protein products not larger than 53 kDa. We term this location of the WD-repeat domains (present in both Sec31B-F latter group of ‘truncated’ proteins Sec31B-T, as represented and Sec31B-T) responsible for binding Sec 13, and the proline- in three of the four transcripts depicted in Fig. 3B. Because the rich domain (>24%) responsible for binding Sec23 (Shaywitz reading frame downstream of the stop codon in exon 13 is et al., 1997). This latter region is deleted in all proteins derived open, we cannot formally exclude the possibility that proteins from Sec31B-T transcripts. also exist that are generated by utilization of initiation sites other than the one in exon 2. Although there is some indication Both Sec 31B-F and Sec31B-T are widely expressed in by western blotting that alternative translation initiation sites tissues and in cultured cells might be utilized (see Fig. 4), we have no conclusive evidence Sec31B expression was evaluated in a variety of tissues and that this is indeed the case. It is also noteworthy that, when cell lines by northern analysis, western blot, and by indirect

Fig. 3. Human Sec31B generates multiple transcripts by alternative mRNA splicing. Three cDNA libraries were examined for alternate exon utilization by PCR. Representative PCR results are shown in the supplementary material Fig. S1. Journal of Cell Science (A) Alternative splicing map of Sec31B representing experimentally observed patterns of exon utilization, based on PCR analysis and EST data available in GenBank. Constitutive exons (green) and exons alternatively utilized (red) are shown along with the frequency of their detection in the examined transcripts. Of the total of 29 exons identified, 16 exons were constitutively present in all transcripts. (B) Examples of four alternatively spliced transcripts depicting their exon utilization. Arrows represent the open reading frames with the predicted molecular mass of the protein products. Although an open reading frame downstream of the stop codon in exon 13 exists, no evidence was identified for the utilization of initiation sites other than that in exon 2. Notice that, if exon 4 is not utilized, then exon 5 contributes an out-of-frame stop codon. (C) Cartoon representing all patterns of exon utilization experimentally identified in this study. Exons encoding WD repeats and proline-rich sequences are indicated. Sec31B alternative transcripts 963

Fig. 4. Sec 31B is widely expressed in tissues and in cultured cells. (A) Northern blots identify the predominant Sec31B mRNA as a series of two or three closely spaced bands centered at 6.5 Kb in all tissues examined, but heavily expressed in testis. Faint bands can also be noticed at approximately 1.0 kb and over 11kb, the later presumably representing traces of genomic contamination. Analysis by dot-blotting of a multiple tissue array confirms abundant expression in testis as well as in cerebellum, thymus, lymph node, pituitary gland and, to a lesser extent, in all tissues on the array. A complete key to the dot-blot array is presented in supplementary material Table S1. (B) Western blots of a series of cultured cell lines using the rabbit IgG 2013 antibody, compared with the same blots probed with the antibody against Sec31A. Preimmune sera shows only a prominent non-specific band at approximately 80 kDa. This non-specific band is barely detectable with the affinity-purified IgG 2013, it demonstrates reactivity with Sec31B-F at 129 kDa as well as with a number of smaller bands. Notice that there is no correspondence of the bands reacting with IgG 2013 and the Sec31A antibody. (C) Cell- and tissue-extracts prepared from rat brain and testis were western blotted with anti-Sec31B antibodies Mab 1G10 and IgY 1871. The control lanes (ctrl) depict the reactivity present in non-immune hybridoma medium against MDCK cells (for Mab 1G10) or in the preimmune IgY (for IgY 1871). Notice the presence of a 129 kDa band with both antibodies (Sec31B-F) and that only Mab 1G10 reacts with a prominent 53 kDa band (Sec31B-T). The star marks the position of the faint non-specific band just below the band for Sec31B-T.

immunoﬂuorescent microscopy. Northern blots

Journal of Cell Science identify the predominant Sec31B mRNA as a series of two or three closely spaced bands centered at 6.5 Kb in all tissues examined, but most abundantly expressed in testis (Fig. 4A). Additional and much fainter bands are also present at approximately 1.0 kb and approximately 11 (or more) kb. Dot blot analysis with a multiple-tissue array demonstrates widespread expression of Sec31B, although the highest levels by far are found in testis and cerebellum (Fig. 4B and supplementary material Table S1 for complete key). To verify translation, we used three antibodies generated against various regions of products of potential transcripts downstream of exon 13. Sec31B to probe extracts of cultured cells and whole testis in Smaller bands can also be noticed. In testis, using the IgY western blots (Fig. 4B,C). The IgG 2013 antibody reacts to 1871 antibody, many similar bands are detected above 80 kDa epitopes encoded by exons 16 and 17. The IgY 1871 antibody that are not in the preimmune sera. Significantly, Mab 1G10 reacts with epitopes encoded by exons 16 to 29. Thus, neither (but not IgY 1871) also detects a prominent protein band at of these antibodies reacts with Sec31B-T. Conversely, Mab 53 kDa slightly above a background band found in hybridoma 1G10 reacts with epitopes found in exons 5 to 11, and medium (Fig. 4C), confirming the expression of Sec31B-T. therefore will detect both Sec31B-F and Sec31B-T (but not The nature of the additional bands on these blots, and whether translation products derived from initiation sites downstream they represent degradation products or the translation products of exon 13, if such products exist). There is no cross-reactivity of other alternatively spliced mRNAs has not been further of these antibodies with the bands detected by an antibody to explored. However, it is noteworthy that recent ESTs Sec31A. In Fig. 4B, ignoring the band at 80 kDa that is in the experimentally identify additional putative transcripts of pre-immune sera, one finds multiple reactive peptides between Sec31B at 45, 38, and 51 kDa, (GenBank AK128343, the full-length Sec31B-F band at 129 kDa and approximately BC044569, BC053949), so it is not unlikely that the 83 kDa that correspond roughly in size to the predicted complexity of alternative splicing and possibly alternative 964 Journal of Cell Science 119 (5)

Fig. 5. Sec31A and Sec31B have distinct distributions in testis and cerebellum. The cellular distribution of Sec31B was compared with Sec31A and Sec13 by indirect immunoﬂuorescence. (A) Comparison with Sec31A. (B) Comparison with Sec13. Notice the concentration of Sec31B- staining in punctate intracellular structures in both Purkinje cells and in Sertoli’s cells. In testis, Sec31B is colocalized with Sec31A and Sec13 predominately at the Golgi region. Unlike Sec31A, there are also coarse cytoplasmic accumulations of Sec31B in the testis. These are less apparent in Purkinje cells.

translation initiation in Sec31B is even greater than what we Three cell lines were examined, NRK cells, MDCK cells, and have so far detected. COS-7 cells; all gave comparable results. The pattern of

Journal of Cell Science The intracellular disposition of Sec31B in testis and Sec31B and Sec31A staining in COS-7 cells is shown in Fig. cerebellum was compared to Sec31A and Sec13 using Mab 6A. Overall, their staining was largely coincident, with 1G10 (Fig. 5). In testis, Sec31B was present in Sertoli’s and concentration of both proteins over the Golgi network, as well Leydig’s cells, in spermatogonia and, less so, in mature as on dispersed small cytoplasmic puncta (Fig. 6A, top panel). sperm. The intracellular distribution of Sec31B was ﬁnely However, they were not 100% coincident, with discrete and granular and diffuse, with concentration over the Golgi region non-overlapping foci of Sec31B or Sec31A apparent. and also in larger, dense cytoplasmic aggregates. It Presumably, the many peripheral small puncta that stained for overlapped the distribution of Sec31A and Sec13 strongly in both Sec31A and Sec31B represent ERESs. However, in some the Golgi region, in the diffuse cytoplasmic pools of the cultured cells the pattern of Sec31B was dominated by (presumably representing ER), but not in the more dense its collection into irregular coarse cytoplasmic aggregates, cytoplasmic accumulations. In the cerebellum, Sec31B was similar to the coarse accumulations observed in testicular expressed in all layers, most prominently in Purkinje and tissue. Because we cannot reliably identify the distribution of granular cells. In Purkinje cells, Sec31B was densely just the truncated forms of endogenous Sec31B in these cells, concentrated into ten to 20 discrete cytoplasmic bodies we explored the behavior of transfected Sec31B-F and (presumably the Golgi network) and, although also diffusely Sec31B-T, expressed as fusions with eYFP (or with eCFP present in the cytoplasm, was not displayed in the same analogues) in COS-7 or MDCK cells. These results are shown abundance as the coarse cytoplasmic aggregates found in the in Fig. 6A (lower panels). Whereas transfected eYFP-Sec31B- testis. By comparison, Sec31A in Purkinje cells displayed a F consistently yielded a pattern indistinguishable from Sec31A prominent diffuse granular cytoplasmic distribution, with (Fig. 6A, middle panel), transfected eYFP-Sec31B-T only concentrations coincident with the staining of Sec31B over partially overlapped the Sec31A staining (Fig. 6A, lower putative Golgi networks. panel). The previous results suggested that Sec31A and Sec31B-F Full-length Sec31B associates with other components of shared the same intracellular distribution. Recent work has the COPII coat indicated that the Sec13-Sec31 complex exists as a The pattern seen in testis and cerebellum, wherein the heterodimer. As a ﬁrst step to determining whether Sec31B-F distribution of Sec31A and Sec31B was partially but not can form mixed dimers directly with Sec31A, lysates from a completely coincident, was also apparent in cultured cells. MDCK cell line stably expressing eYFP-Sec31B-F (MDCK- Sec31B alternative transcripts 965

Fig. 6. Sec31B-F and Sec31A share the same intracellular distribution. (A) The distribution of full-length and truncated Sec31B was compared with endogenous Sec31A in Cos-7 cells. In all panels, endogenous wild-type Sec31A is stained red; Sec31B (wild-type or transfected) is stained green. Areas of coincidence appear yellow. (Top) Comparison of the endogenous proteins; overlap is extensive, but not complete. (Middle) Comparison of wt Sec31A with transfected eGYP-Sec31B- F; overlap is essentially complete. (Bottom) Comparison of wt Sec31A with transfected eGYP-Sec31B-T; notice the absence of any overlap. Arrows indicate discrete non-overlapping accumulations of Sec31A and Sec31B. (B) Lysates of the MDCK-787 cell line that stably expresses eYFP-Sec31B-F were immunoprecipitated with the antibody against Sec31A (kindly supplied by Fred Gorlick, Yale University). The precipitates were then probed for the presence of either eYFP-Sec31B-F (left panel) or for Sec31A (right panel). Non- immunesera was used as the control (ctrl). Notice that anti-Sec31A does not cross- react with eYFP-Sec31B-F.

787) were immunoprecipitated with antibodies against Sec31A, and then western blotted to detect eYFP-Sec31B-F. These studies detected a clear and direct association between Sec31A and Sec31B-F (Fig. 6B).

Journal of Cell Science The association of Sec31B with two other components of the COPII coat, Sec23 and Sec13, was also explored. Again, in native COS-7 cells, there was only partial overlap between Sec31B and Sec23 (Fig. 7A, top panel). As with the comparison with Sec31A, transfected eYFP-Sec31B-F demonstrated nearly complete overlap with Sec23, whereas transfected eYFP-Sec31B-T demonstrated no coincident the peptide encompassing the WD repeats bound Sec13 in staining. The close association of eYFP-Sec31B-F with Sec23 MDCK cell lysates (Fig. 7B). was further revealed by double-label electron microscopy (EM) (Fig. 7A). Both proteins were intermingled and Sec31B-F displays pharmacologic and dynamic coincident at the EM level, on both vesicular tubular clusters behaviors that conﬁrm its participation with COPII- (VTCs) and on some 50-60-nm vesicles. coated organelles The association of Sec31B-F with Sec13 was detected by To further characterize the association of Sec31B-F with the coincident staining in cultured cells, by earliest steps in the secretory pathway, its distribution, response coimmunoprecipitation from native MDCK cells, and by GST- to blockage of ERESs or microtubule disruption, and its pull-down assays from MDCK cell lysates (Fig. 7B). The intracellular dynamics of motion were investigated. Incubation immunoﬂuorescent results shown in Fig. 7 are from cells of cells at low temperature allows the accumulation of COPII- incubated at 15°C to block the release of COPII vesicles from coated exit complexes along the ER, but prevents completion of the ERES. There is almost perfect coincidence of Sec31B-F their budding- and release-cycle. The MDCK-787 line was with Sec13 staining at the putative ERES. transfected with eCFP-VSV-G-tsO45, and the distribution of Immunoprecipitation from the MDCK-787 line expressing eYFP-Sec31B-F, eCFP-VSV-G-tsO45 and Sec13 were eYFP-Sec31B-F with anti-GFP antibody co-precipitated compared 4 minutes after the cells were returned to RT from a Sec13. The region of Sec31B responsible for binding Sec13 15°C incubation. Under these conditions, a large number of was examined with GST-fusion peptides representing, the N- ts045-VSV-G-CFP (VSV-G)-loaded and Sec13-positive VTCs terminal region of Sec31B with its WD repeats (residues 161 are apparent. These are highlighted in Fig. 8A by the small to 471), or a GST-fusion peptide (residues 818 to 1172) from arrows. Essentially, all of these clusters contain Sec31B-F. In an the C-terminal region including its proline-rich domain. Only alternative approach, the same cell line expressing eCFP-VSV- 966 Journal of Cell Science 119 (5)

Fig. 7. Sec31B interacts with Sec13 and Sec23. (A) The distribution of full-length and truncated Sec31B was compared with endogenous Sec23 in COS-7 and MDCK cells. Fluorescent mages from the COS-7 cells are shown. In all panels, endogenous wt Sec23 is stained red; Sec31B (wild-type or transfected) is stained green. Areas of coincidence appear yellow. (Top and middle rows) Comparison of the endogenous proteins Journal of Cell Science or with transfected eYFP-Sec31B-F; overlap is extensive. (Bottom) Comparison of wild-type Sec23 with transfected eYFP-Sec31B-T; notice the absence of overlap. (EM) Thin sections of MDCK-787 cells embedded in LR white were stained with colloidal gold for Sec23 (5 nm particles) and for eYFP-Sec31B-F (10 nm particles). Notice the close association of staining over regions of vesicular tubular clusters (vtc) and on some 50-60 nm vesicles indicated by *. There is no staining of the Golgi cisternae (G). (B) Comparison of the distribution of endogenous Sec13 and eYFP-Sec31B-F in MDCK-787 cells that were also transfected with VSV-G, ﬁve minutes after incubation at permissive temperature (32°C). Staining of VSV-G is not shown, it was coincident with Sec13. Notice the perfect association of Sec13 staining with VSV-G in the punctate ERESs. (IP) Immunoprecipitation of the MDCK-787 cells with antibodies against GFP (Sec31B) or Sec13 co-precipitate Sec13. Non- immune sera (ctrl); cell lysates (lys). (GST pull-down) The direct interaction between Sec31B and Sec13 is mediated by the WD-repeat domain of Sec31B because a GST-fusion peptide encompassing the WD region of Sec31B efficiently binds Sec13 in MDCK cell lysates (WD), whereas a similar fusion protein derived from the C-terminal proline-rich domain of Sec31B (PRD) that is devoid of the WD region does not bind Sec13. Ctrl, GST alone; lys, MDCK cell lysate used in the pull-down assay.

G-tsO45 was treated with nocodazole to disrupt their Consistent with its role as a COPII-coat component, eYFP- microtubules, under conditions in which the eCFP-VSV-G- Sec31B-F was associated only briefly with VSV-G during its tsO45 could enter the secretory pathway after a brief incubation exit from the ER, and then remained relatively stationary as at a permissive temperature (Fig. 8B). Sec31B-F clumped into VSV-G moved towards the Golgi network (arrows in Fig. 9). coarse punctate clusters dispersed throughout the cell, consistent In the supplementary material, Movies 1-3 illustrate the with the behavior of other COPII components. The punctate motion of eYFP-Sec31B-F; and several additional figures clusters were loaded with VSV-G at permissive temperature. (supplementary material Figs S2-S5) depict the intracellular Finally, the dynamic motion of Sec31B-F was examined and distribution of Sec31B-F under a variety of conditions. compared to the transport of tsO45-VSV-G-CFP in transfected COS-7 cells (Fig. 9). Whereas cargo laden vesicles exiting the Discussion ER undergo rapid long-range movement towards the Golgi A novel mammalian orthologue of the yeast protein Sec31p has network along microtubule pathways (Cole and Lippincott- been cloned and characterized. This protein, termed Sec31B, Schwartz, 1995), COPII complexes typically remain tightly arises from a 33 kb gene composed of 29 exons. Multiple associated with the ER, and their movement is confined to isoforms of Sec31B are generated by alternative mRNA splicing within one or two microns of ERESs (Stephens et al., 2000). and these isoforms fall into two distinct categories. When exon Sec31B alternative transcripts 967

13 is not utilized, one – of several – full-length proteins generates a family of truncated proteins (one which is Sec31B- (Sec31B-F) is translated that functions as a conventional COPII T). These truncated proteins appear to associate with the ER but component. When exon 13 is utilized, its in-frame stop codon not in the same way as the full-length Sec31 proteins. These

Fig. 8. Sec31B-F moves with COPII complexes at different temperature or after disruption with nocodazole. The MDCK-787 cells expressing eYFP- Sec31B-F were transfected with VSV- G, and the dynamics of these proteins was measured under conditions of low-temperature blockage of ER export, or nocodazole disruption of microtubule-based intracellular transport. (A). Cells blocked at 15°C were warmed to room temperature (RT) for 4 minutes to allow entry of VSV-G into the ERESs. Cells were then ﬁxed and examined for the above proteins. Under these conditions, VSV-G clusters into hundreds of minute ERESs that are coincident with COPII vesicular tubular clusters as detected by their Sec13 staining. The distribution of eYFP-Sec31B-F was perfectly coincident with these COPII-enriched ERESs. (B) Similar preparation of MDCK-787 cells blocked with nocodazole and incubated for 15 minutes at RT after a 2 hour incubation at 40°C. Under these conditions, eYFP-Sec31B-F forms coarse puncated clustes closely associated with the dispersed Golgi network to which VSV-G had moved during the 15 minute incubation at the permissive temperature (RT). Bars, 10 ␮m. Journal of Cell Science

Fig. 9. VSV-G exiting the ER transiently associates with Sec31B-F. COS-7 cells were transiently transfected with VSV-G (construct 472, green) and eYFP-Sec31B-F (construct 787, yellow), colored red. After accumulating VSV-G in the ER for 3 hours at 40°C, cells were shifted to a permissive temperature (32°C) and images collected every 12 seconds over a period of 10 minutes. A selection of images at the times indicated is shown. The VSV-G can be seen ﬁlling the Golgi network. Also visible are many small transport vesicles exiting the ER and moving towards the Golgi network. The eYFP-Sec31B-F oscillates in the vicinity of ERESs but does not move towards the Golgi network. The insert, magniﬁed 4ϫ, demonstrates VSV-G and eYFP-Sec31B-F temporarily merged at the ERES; after 24 seconds the VSV-G vesicle moves anterograde towards the Golgi network, while eYFP-Sec31B-F stays behind. Arrows indicate the locus of a Sec31B-F vesicle (red) and a VSV- G-loaded vesicle (green). Bar, 10 ␮m. 968 Journal of Cell Science 119 (5)

tools from DNAStar Inc. The intron-exon boundaries were established by conclusions are supported by several lines of evidence: (1) The comparing the entire cDNA sequence against the sequence of chromosome 10, Sec31B gene shows strong homology with both yeast Sec31p followed by heuristic inspection of the resulting pair-wise comparison for canonical and mammalian Sec31A. (2) Multiple transcripts displaying splice junctions. alternative exon utilization have been generated by PCR from Northern analysis both brain and testis cDNA libraries. (3) Published ESTs confirm Using a gel-purified fragment (bp 1331-2579) random labeled with [32P] dATP, a the cloning and PCR data indicating multiple alternative human normal mRNA Blot IV (Invitrogen; catalog number D1804-08) was probed transcripts. (4) Western blotting with three independently following the manufacturer’s instructions. The blot was then stripped and probed generated and affinity-purified antibodies confirms both for actin transcripts to confirm equal loading of housekeeping genes. Similarly, a labeled cDNA probe from bps 1384-3670 was used with a Human Multiple Tissue Sec31B-F and Sec31B-T transcripts in a variety of cell lines, and Express Array (Catalog number 7775-1, Clontech, Palo Alto) to examine a wider in testis extracts, as well as the presence of multiple tissue distribution. immunoreactive bands consistent with many of the predicted alternative transcripts. (5) Brain and testis tissues and cultured Production of prokaryotic fusion proteins and eukaryotic expression vectors cells display partially coincident staining for Sec31A, Sec13 and Cloning of the N-terminal region of Sec31B containing the WD motif was Sec31B. (6) Transfected GFP-tagged Sec31B-F and Sec31B-T accomplished by PCR using oligonucleotides 51853 and 51865 (supplementary do not exactly colocalize in cultured cells. (7) Sec31B-F binds material Fig. S1). PCR products were gel-purified from low-melt agarose (FMC) using Pharmacia GFX gel purification kit, digested with EcoRI and cloned into Sec13 through its WD-repeat domains, and co-distributes with pGEX-4T1 (Pharmacia). Expression of fusion protein occurred in DH5␣ bacteria other COPII markers under all conditions examined. (8) after the addition of 0.1 mM IPTG. Cloning of the C-terminal proline-rich domain Sec31B-F and Sec23 are coincident on 50-60 nm vesicles and was accomplished in a similar way using oligonucleotides 48827 and 49372, except VTCs by immunoelectronmicroscopy. (9) Sec31B-F displays that the PCR product was digested with both EcoRI and BamHI to take advantage of the internal BamHI site located within the amplimer. Full-length Sec31B-F was restricted intracellular movement, remaining in close proximity assembled into the XhoI and AccI sites of pEGFP-3n (BD Biosciences) using to putative ERESs whereas VSV-G moves rapidly away from sequence-verified PCR amplimers. During this ligation the AccI site was destroyed these sites to the Golgi network, a dynamic identical to that of after annealing with the compatible overhang of a ClaI site, part of sense oligo 63730 used to amplify Sec31B. This construct was then digested with SmaI and DraIII and another COPII component, Sec23/24 (Stephens et al., 2000). Not the GFP part of the vector replaced with a yellow fluorescent protein from pEYFP- addressed in this study is the role of the truncated isoforms of 1N (BD Biosciences). The truncated version Sec31B-T was constructed in a similar Sec31B, i.e. Sec31B-T and possibly other short isoforms arising way by ligating a PCR amplimer, which contained the endogenous stop codon from from alternative translation initiation sites that are hinted at by exon 13, into the XhoI and KpnI sites of pEYFP-C1 (BD Biosciences). some of the western blot data. Creation of the stable MDCK-787 cell line expressing eYFP- Presumably, the presence of Sec31B-F bestows on a cell the Sec31B-F capacity to generate a COPII coat with slightly modified The cDNA for eYFP-Sec31B-F (787) was transfected into MDCK cells using dynamics or cargo specificities. It also appears probable that, Lipofectamine 2000 following the manufacturer’s instructions. After 48 hours the cells were plated at dilutions into Dulbecco’s minimal Eagle’s medium (DMEM) based on the ability of Sec31B-F to coimmunoprecipitate with supplemented with 400 ␮g/ml of G418 (Geneticin, Invitrogen). After three weeks, Sec31A (as well as with Sec13) and given recent indications G418-resistant colonies were subcloned and screened by fluorescent microscopy for that the functional Sec13/31 complex is a heterotetramer the expression of eYFP. Positive clones were expanded and tested by western blot using an anti-GFP antibody for the expression of a single 158 kDa band. Four (Lederkremer et al., 2001), COPII-coat structures must exist, independent colonies exhibiting the best expression data were chosen for use in Journal of Cell Science which not only contain various transcripts of Sec31B-F but also these studies. All gave identical results. mixed heterotetramers of Sec13-Sec31B and Sec13-Sec31A. These results thus raise for the first time the intriguing Antibody preparation and immunologic procedures Three antibodies were prepared for this study. An IgY antibody (IgY1871) (Aves possibility that the Sec13-Sec31 cage, like Sec23-Sec24, is built Labs, Tigard, OR) against a recombinant peptide generated by expressing cDNA with isoforms tuned for specific types of cargo. Examples of corresponding to residues 818-1172, exons 16 to 29, in pGEX-3T (Amersham such might include those reported for procollagen (Mironov et Pharmacia Biotech) was prepared in chickens. This antibody was affinity-purified by desorption from Millipore Immobilon-P membranes loaded with the GST-tagged al., 2003), chylomicrons (Siddiqi et al., 2003) or GPI-linked Sec31B peptide. Affinity purified antibodies were eluted from Immobilon-P proteins (Muniz et al., 2001). In future work, it will be important membranes with 200 mM glycine, 500 mM NaCl pH 2.8, and neutralized with 1 M to explore not only the potential role(s) of the truncated forms Tris pH 8.0. The second antibody prepared was a mouse monoclonal antibody of Sec31B, but also whether the efficient export of different (Mab). Two independent Mab clones (Mab 1D4 and 1G10) (Maine Biotechnologies) were identified against a GST-fusion peptide derived from exons 5-13 of Sec31B. cargos requires different combinations of Sec31B isoforms and The third antibody was an affinity-purified rabbit IgG (IgG2013) against a synthetic Sec31A. If so, such a finding might explain the differential peptide bridging exons 16 and 17 of Sec31B. The peptide sequence appearance of Sec31B seen in different tissues. MTPWEIPITKDIDGL corresponds to codons 552-566. We chose this peptide because it shares little homology to Sec31A or Sec31p. All antibodies were evaluated for specificity by: (i) comparison with preimmune sera; (ii) by competitive Materials and Methods inhibition of western blots by the recombinant or synthetic peptide used to generate Cloning and sequencing the antibody; and (iii) by confirmation of specificity using western blots of cell All molecular biological procedures followed standard methods (Maniatis et al., extracts overexpressing the recombinant Sec31B peptides as eYFP-fusion proteins. 1982) unless otherwise stated. Candidate sequences were derived from GenBank by The antibody directed against GFP was from Clontech, the Sec23 antibody was a TBLASTN searches using sequences from rat Sec31A (GenBank accession number rabbit IgG from Affinity Bioreagents. Other antibodies were obtained from private AF034582). Oligonucleotides were designed to amplify the entire Sec31B cDNA sources as indicated in the acknowledgements. from ClontechTM human testis, brain or fetal brain Marathon Ready cDNA libraries using Platinum Taq high fidelity polymerase from Invitrogen Corporation, following Immunocytochemistry manufacturer’s protocols. Primary and nested PCR, 5Ј RACE, TA-TOPO cloning COS-7 and MDCK cells were grown in Eagle’s or DMEM with 10% fetal BSA on and sequencing were conducted as before (Stabach and Morrow, 2000). The Falcon tissue culture chamber slides. Both wild type cells and cells transfected by oligonucleotides used in this study are listed in supplemental Figure S1. To Lipofectamine 2000 (Invitrogen) were fixed with either 50:50 v/v mixture of eliminate errors associated with amplification, multiple overlapping PCR reaction acetone/methanol at –20°C for 10 minutes; permeabilized with 0.1% saponin in products were cloned and sequenced over the entire length of Sec31B and PBS; and incubated with Mabs 1G10 or 1G10 diluted 1:50 in PBS with 0.1% compared, to the dbESTs from GenBank and the sequence of chromosome 10 (clone saponin, 2% BSA (Fraction V; Sigma Chemical Co., St Louis, MO). Cells were RP11-411B6) deposited from the Sanger Centre. Sequence analysis was performed washed with PBS, and incubated with 1:1000 secondary antibody conjugated to using software from Gene Construction Kit, Textco Inc., and a portfolio of analysis Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes). Sec31B alternative transcripts 969

Electron microscopy ankyrin G119 skeleton in Madin Darby canine kidney cells. Proc. Natl. Acad. Sci. USA Cells grown on 10 cm plastic Petri dishes were fixed overnight with a mixture of 94, 10711-10716. 4% formaldehyde and 0.1% glutaraldehyde in PBS pH 7.4. Cells were collected and Klumperman, J. (2000). Transport between ER and Golgi. Curr. Opin. Cell Biol. 12, pelleted, and cell pellets were washed with PBS, dehydrated on ice in graded ethanol 445-449. before LR White resin-infiltration. Resin was polymerized in sealed gelatin capsules Kuge, O., Dascher, C., Orci, L., Rowe, T., Amherdt, M., Plutner, H., Ravazzola, M., at 50°C for 48 hours. Thin sections were cut and adhered to carbon-coated formvar Tanigawa, G., Rothman, J. E. and Balch, W. E. (1994). Sar1 promotes vesicle grids. Gold labeling was performed with anti-GFP (Clontech), and anti Sec-23 budding from the endoplasmic reticulum but not Golgi compartments. J. Cell Biol. 125, (ABR) antibodies. Thin sections were blocked with 1% BSA, 0.1M glycine in PBS, 51-65. Lederkremer, G. Z., Cheng, Y., Petre, B. M., Vogan, E., Springer, S., Schekman, R., incubated with Sec23 antibody for 2 hours followed by 1 hour incubation with 5 Walz, T. and Kirchhausen, T. (2001). Structure of the Sec23p/24p and Sec13p/31p nm goat anti-rabbit colloidal gold. Cells were re-fixed with 1% glutaraldehyde, re- complexes of COPII. Proc. Natl. Acad. Sci. USA 98, 10704-10709. blocked and labeled with GFP antibody followed by 10 nm goat anti-rabbit colloidal Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982). Molecular Cloning: A Laboratory gold. Sections were then re-fixed in 1% glutaraldehyde, PBS washed, air-dried and Manual. New York: Cold Spring Harbor Laboratory. post stained with 0.5% uranyl acetate and 1 mg/ml lead citrate. Sections were Matsuoka, K., Morimitsu, Y., Uchida, K. and Schekman, R. (1998). Coat assembly examined and imaged on a Zeiss EM-910 microscope at 80 KV. directs v-SNARE concentration into synthetic COPII vesicles. Mol. Cell 2, 703-708. Matsuoka, K., Schekman, R., Orci, L. and Heuser, J. E. (2001). Surface structure of ts045-VSV-G-eCFP (VSV-G)-transport studies the COPII-coated vesicle. Proc. Natl. Acad. Sci. USA 98, 13705-13709. The ts045-VSV-G-eCFP (VSV-G) construct was generated from a ts045-VSV-G- Miller, E. A., Beilharz, T. H., Malkus, P. N., Lee, M. C., Hamamoto, S., Orci, L. and GFP clone (a gift from J. Lippincott-Schwartz, NIH) by replacement of the GFP Schekman, R. (2003). Multiple cargo binding sites on the COPII subunit Sec24p with the enhanced cyan fluorescent protein (eCFP) derived from pECFP-1N (BD ensure capture of diverse membrane proteins into transport vesicles. Cell 114, 497- Biosciences). COS-7 cells transiently co-transfected with full-length eYFP-Sec31B 509. (or with truncated variants) and VSV-G, were incubated at the non-permissive Mironov, A. A., Mironov, A. A., Jr, Beznoussenko, G. V., Trucco, A., Lupetti, P., temperature of 40°C for 3-6 hours to load the ER with newly translated VSV-G, Smith, J. D., Geerts, W. J., Koster, A. J., Burger, K. N., Martone, M. E. et al. (2003). ER-to-Golgi carriers arise through direct en bloc protrusion and multistage and then shifted to either 15°C (to block exit at the ERES) or 32°C to study the maturation of specialized ER exit domains. Dev. Cell 5, 583-594. kinetics of synchronized transport of VSV-G to the Golgi network. Fluorescence Mossessova, E., Bickford, L. C. and Goldberg, J. (2003). SNARE selectivity of the microscopy of the cells was done with an Olympus IX70 dual laser scanning TM COPII coat. Cell 114, 483-495. confocal microscope, or with an Olympus AX70 equipped with Openlab Muniz, M., Morsomme, P. and Riezman, H. (2001). Protein sorting upon exit from the (Improvision, Lexington, MA) deconvolution software. Time-lapse vital cell endoplasmic reticulum. Cell 104, 313-320. microscopy was done with cells grown on glass coverslips and housed in a flow- Paccaud, J. P., Reith, W., Carpentier, J. L., Ravazzola, M., Amherdt, M., Schekman, cell-containing growth medium. These were imaged using an Olympus Till R. and Orci, L. (1996). Cloning and functional characterization of mammalian Photonics time-lapse microscope (Olympus IX70) with TILL Imago CCD camera homologues of the COPII component Sec23. Mol. Biol. Cell 7, 1535-1546. and Polychrome II multiwavelength illumination system, all controlled by TILL Pagano, A., Letourneur, F., Garcia-Estefania, D., Carpentier, J. L., Orci, L. and vision software (TILL Photonics LLC, Pleasanton, CA). Paccaud, J. P. (1999). Sec24 proteins and sorting at the endoplasmic reticulum. J. Biol. Chem. 274, 7833-7840. Other procedures Penalver, E., Lucero, P., Moreno, E. and Lagunas, R. (1999). Clathrin and two The interaction of the recombinant Sec31B peptides with Sec13 was determined by components of the COPII complex, Sec23p and Sec24p, could be involved in the ability of GST-Sec31B peptides to capture Sec13 from MDCK cell extracts in endocytosis of the Saccharomyces cerevisiae maltose transporter. J. Bacteriol. 181, 2555-2563. pull-down assays or by coimmunoprecipitation. MDCK cells were extracted with a Pradhan, D. and Morrow, J. S. (2002). The spectrin-ankyrin skeleton controls CD45 nondenaturing buffer (20 mM Hepes pH 7.4, 120 mM NaCl, 25 mM KCl, 2 mM surface display and interleukin-2 production. Immunity 17, 303-315. EDTA, 1 mM EGTA, 0.1% Triton X-100). Typically, 0.1 mg of GST-Sec31B peptide Salama, N. R., Chuang, J. S. and Schekman, R. W. (1997). Sec31 encodes an essential in 50 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT would be absorbed component of the COPII coat required for transport vesicle budding from the ␮ to 50 l of 1:1 slurry of glutathione-Sepharose. Proteins retained after three washes endoplasmic reticulum. Mol. Biol. Cell 8, 205-217. in the same buffer were analyzed by western blot after SDS-PAGE. For Shaywitz, D. A., Orci, L., Ravazzola, M., Swaroop, A. and Kaiser, C. A. (1995). immunoprecipitation experiments, MDCK cell extracts were pre-cleared with Human SEC13Rp functions in yeast and is located on transport vesicles budding from Protein A-tris acryl (Pierce), incubated overnight at 4°C with primary antibodies the endoplasmic reticulum. J. Cell Biol. 128, 769-777. and proteins captured with Protein A-tris acryl. Proteins retained on the resin after Shaywitz, D. A., Espenshade, P. J., Gimeno, R. E. and Kaiser, C. A. (1997). 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