Fukuda M, Mikoshiba K
Developmental Neurobiology Laboratory, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Biol Chem. 1999 Oct 29;274(44):31428-34. doi: 10.1074/jbc.274.44.31428.
Synaptotagmins are a family of membrane proteins that are characterized by a single transmembrane region and tandem C2 domains and that are likely to regulate constitutive and/or regulated vesicle traffic. We have shown that a subclass of synaptotagmins (III, V, VI, and X) forms homo- and heterodimers through an evolutionarily conserved cysteine motif at their N termini (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). In this study, we identified a novel alternatively spliced variant of synaptotagmin (Syt) VI that lacks the N-terminal 85 amino acids including the transmembrane region (thus designated as Syt VIDeltaTM). Because it lacks the cysteine motif responsible for self-dimerization, Syt VIDeltaTM could not associate with Syt VI even in the presence of Ca(2+). Despite lacking the transmembrane region, Syt VIDeltaTM can associate with the plasma membrane through the C-terminal 29 amino acids. In adult mouse brain, two closely comigrating bands at M(r) approximately 50,000, which closely corresponded to the molecular weight of recombinant Syt VIDeltaTM, were detected by anti-Syt VI antibody. These immunoreactive bands were found in both soluble and membrane fractions of mouse brain, indicating that they are membrane-associated proteins (Syt VIDeltaTM), but not transmembrane proteins (Syt VI). Expression of Syt VI and Syt VIDeltaTM in PC12 or COS-7 cells indicated that the two molecules have a distinct subcellular distribution: Syt VIDeltaTM is present in the cytosol or is associated with the plasma membrane or internal membrane structures, whereas Syt VI is localized to the endoplasmic reticulum and/or Golgi-like perinuclear compartment. These results suggest that Syt VI and Syt VIDeltaTM may play distinct roles in vesicular trafficking.
突触结合蛋白是一类膜蛋白，其特征在于具有单个跨膜区域和串联的C2结构域，并且可能调节组成型和/或调节型囊泡运输。我们已经表明，突触结合蛋白的一个亚类（III、V、VI和X）通过其N端进化保守的半胱氨酸基序形成同二聚体和异二聚体（福田，M.，神野，E.，和三室，K.（1999年）《生物化学杂志》274，31421 - 31427）。在本研究中，我们鉴定出一种新的突触结合蛋白（Syt）VI的可变剪接变体，其缺少包括跨膜区域在内的N端85个氨基酸（因此命名为Syt VIDeltaTM）。由于它缺少负责自身二聚化的半胱氨酸基序，即使在存在Ca(2+)的情况下，Syt VIDeltaTM也不能与Syt VI结合。尽管缺少跨膜区域，Syt VIDeltaTM仍可通过C端的29个氨基酸与质膜结合。在成年小鼠脑中，抗Syt VI抗体检测到两条紧密共迁移的条带，分子量约为50,000，这与重组Syt VIDeltaTM的分子量密切对应。这些免疫反应性条带出现在小鼠脑的可溶性和膜部分中，表明它们是膜相关蛋白（Syt VIDeltaTM），而不是跨膜蛋白（Syt VI）。Syt VI和Syt VIDeltaTM在PC12或COS - 7细胞中的表达表明这两种分子具有不同的亚细胞分布：Syt VIDeltaTM存在于细胞质中，或与质膜或内膜结构相关，而Syt VI定位于内质网和/或高尔基体样核周区室。这些结果表明Syt VI和Syt VIDeltaTM可能在囊泡运输中发挥不同的作用。
