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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02751

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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

View larger version (97K): [in a new window]   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).

View larger version (66K): [in a new window]   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 5' 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.

View larger version (28K): [in a new window]   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. (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.

View larger version (60K): [in a new window]   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 pre-immune 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.

View larger version (96K): [in a new window]   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.

View larger version (52K): [in a new window]   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.

View larger version (59K): [in a new window]   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 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.

View larger version (37K): [in a new window]   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 µm.

View larger version (61K): [in a new window]   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 4x, 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 µm.

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