Stankewich Michael C, Stabach Paul R, Morrow Jon S
Department of Pathology, Yale University, New Haven, CT 06520, USA.
J Cell Sci. 2006 Mar 1;119(Pt 5):958-69. doi: 10.1242/jcs.02751.
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.
我们克隆了人类大脑和睾丸中的Sec31B蛋白（也称为分泌途径组分Sec31B-1或类Sec31 2；GenBank登录号AF274863）。Sec31B是酿酒酵母Sec31p的直系同源物，Sec31p是COPII囊泡衣被的一个组分，介导从内质网的囊泡运输。Sec31B广泛表达，并在小脑和睾丸中富集。其预测的1180个残基序列（预期分子量128,711 Da）与人Sec31A（也称为Sec31；GenBank登录号AF139184）和酵母Sec31p分别具有47.3%和18.8%的相似性。编码Sec31B的基因位于10号染色体q24上，包含29个外显子。对外显子利用的PCR分析揭示了Sec31B存在大量可变mRNA剪接，所有转录本中仅有16个外显子是组成型使用的。外显子13中存在一个终止密码子产生了两个Sec31B基因产物家族（每个家族还显示出额外的mRNA剪接模式）：一组全长蛋白（以下称为Sec31B-F）和一组截短蛋白（以下称为Sec31B-T），它们通过对外显子13的使用而区分。Sec31B-F与Sec31p和Sec31A非常相似，在结合Sec13的N端结构域中有典型的WD重复序列，以及一个可能结合Sec23/24的富含脯氨酸的C端区域。Sec31B-T组（分子量52,983 Da）包含一个保留的WD重复结构域，但缺少C端富含脯氨酸的区域。当在培养细胞中作为与eYFP的融合蛋白表达时，Sec31B-F与内质网和囊泡管状簇相关联，表现出COPII囊泡动力学特有的受限细胞内运动，与Sec13、Sec31A和Sec23（COPII衣被的标志物）在细胞器上共同分布，并且在分泌途径早期或温度或诺考达唑抑制后检查时与ts045-VSV-G-CFP（VSV-G）共聚集。截短形式的Sec31B-T的作用似乎与Sec31B-F不同，仍然未知。我们得出结论，Sec31B-F有助于哺乳动物COPII衣被的多样性，并推测Sec31笼状结构，与Sec24一样，可能是由针对特定类型货物或其他特殊功能进行调节的异构体构建而成。
