Abstract
We have cloned human brain and testis Sec31B protein (also known as secretory pathway component Sec31B-1 or SEC31-like 2; GenBank accession number AF274863). Sec31B is an orthologue of Saccharomyces cerevisiae Sec31p, a component of the COPII vesicle coat that mediates vesicular traffic from the endoplasmic reticulum. Sec31B is widely expressed and enriched in cerebellum and testis. Its predicted sequence of 1180 residues (expected molecular mass 128,711 Da) shares 47.3% and 18.8% similarity to human Sec31A (also known as Sec31; GenBank accession number AF139184) and yeast Sec31p, respectively. The gene encoding Sec31B is located on chromosome 10q24 and contains 29 exons. PCR analysis of exon utilization reveals massive alternative mRNA splicing of Sec31B, with just 16 exons being constitutively utilized in all transcripts. The presence of a stop codon in exon 13 generates two families of Sec31B gene products (each displaying additional patterns of mRNA splicing): a group of full-length proteins (hereafter referred to as Sec31B-F) and also a group of truncated proteins (hereafter referred to as Sec31B-T), distinguished by their utilization of exon 13. Sec31B-F closely resembles Sec31p and Sec31A, with canonical WD repeats in an N-terminal domain that binds Sec13 and a proline-rich C-terminal region that presumably binds Sec23/24. The Sec31B-T group (molecular mass 52,983 Da) contains a preserved WD-repeat domain but lacks the C-terminal proline-rich region. When expressed as a fusion protein with eYFP in cultured cells, Sec31B-F associates with the endoplasmic reticulum and with vesicular-tubular clusters, displays restricted intracellular movement characteristic of COPII vesicle dynamics, co-distributes on organelles with Sec13, Sec31A and Sec23 (markers of the COPII coat), and concentrates with ts045-VSV-G-CFP (VSV-G) when examined early in the secretory pathway or after temperature or nocodazole inhibition. The role of the truncated form Sec31B-T appears to be distinct from that of Sec31B-F and remains unknown. We conclude that Sec31B-F contributes to the diversity of the mammalian COPII coat, and speculate that the Sec31 cage, like Sec24, might be built with isoforms tuned to specific types of cargo or to other specialized functions.
Highlights
Entry to the secretory pathway is initiated by the extrusion of COPII-coated cargo-loaded transport vesicles and tubules from the endoplasmic reticulum (ER)
The packaging of procollagen, a molecule too large to fit into a 60-85 nM COPII vesicle, appears to exit the ER through en bloc protrusion of a patch of ER membrane that is devoid of a COPII coat (Mironov et al, 2003)
In addition to being widely expressed in mammalian tissues, mRNA transcripts of Sec31B display considerable alternative mRNA splicing, generating a range of highly divergent protein products that fall into two general categories, several alternative versions of full-length Sec31B proteins with a mass of about 129 kDa, and a group of truncated proteins including one of about 53 kDa
Summary
Entry to the secretory pathway is initiated by the extrusion of COPII-coated cargo-loaded transport vesicles and tubules from the endoplasmic reticulum (ER). Cytoskeletal proteins that are absent in yeast, play a role in mammalian cells in determining the efficiency of transport of selected proteins through the secretory pathway, including even the efficiency of their exit from the ER and their pathway of intracellular targeting and/or retention (Devarajan et al, 1997; Tuvia et al, 1999; De Matteis and Morrow, 2000; Pradhan and Morrow, 2002) Such findings suggest that evolution has modified and added to the basic ER-exit and -sorting formula found in yeast, to meet the more complex and specialized needs of higher organisms. Both mechanisms appear to have been used in refining the mechanisms of ER export
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