AtVPS45 Complex Formation at the trans-Golgi Network

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide–sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.

Sec1p蛋白家族（Sec1p family of proteins）被认为通过与靶膜上的t-SNAREs（靶可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体，target soluble N-ethylmaleimide–sensitive factor attachment protein receptors）相互作用，参与囊泡融合反应的调控。AtVPS45是拟南芥（Arabidopsis thaliana）来源的该家族成员，我们现已证实其定位于反式高尔基体网络（trans-Golgi network，TGN），并与液泡货物受体AtELP共定位。与酵母Vps45p不同，AtVPS45既不与前液泡t-SNARE AtPEP12相互作用，也不与其共定位。相反，AtVPS45可与两种t-SNARE——AtTLG2a和AtTLG2b——相互作用，这两种蛋白与酵母t-SNARE Tlg2p具有序列相似性。AtTLG2a和AtTLG2b均与AtVPS45共定位于反式高尔基体网络；但通过免疫金电子显微镜观察发现，AtTLG2a与AtTLG2b在反式高尔基体网络中处于不同的亚区域。因此，我们提出包含AtVPS45与AtTLG2a或AtTLG2b的复合物，定义了反式高尔基体网络的功能性亚结构域，且可能参与不同的囊泡运输事件。在其他拟南芥SNARE蛋白中，AtVPS45抗体优先共沉淀同源性较高的AtVTI1b亚型，而非AtVTI1a，这表明AtVTI1a与AtVTI1b在细胞内也具有不同的功能。上述数据表明，植物分泌通路中存在功能复杂性：由基因家族编码的蛋白往往具有特化的功能，而非功能冗余。