Hasegawa Haruki, Yang Zhifen, Oltedal Leif, Davanger Svend, Hay Jesse C
University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI 48109-1048, USA.
J Cell Sci. 2004 Sep 1;117(Pt 19):4495-508. doi: 10.1242/jcs.01314.
Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life. The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons. Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function. Targeting of Ykt6 to its unique subcellular location was directed by its profilin-like longin domain. We have taken advantage of high-resolution structural data available for the yeast Ykt6p longin domain to examine mechanisms by which the mammalian longin domain controls Ykt6 conformation and subcellular targeting. We found that the overall tertiary structure of the longin domain, not sequence-specific surface features, drives direct targeting to the Ykt6 punctate structures. However, several sequence-specific surface features of the longin domain indirectly regulate Ykt6 localization through intramolecular interactions that mask otherwise-dominant targeting signals on the SNARE motif and lipid groups. Specifically, two hydrophobic binding pockets, one on each face of the longin domain, and one mixed hydrophobic/charged surface, participate in protein-protein interactions with the SNARE motif and protein-lipid interactions with the lipid group(s) at the molecule's C-terminus. One of the hydrophobic pockets suppresses protein-palmitoylation-dependent mislocalization of Ykt6 to the plasma membrane. The Ykt6 intramolecular interactions would be predicted to create a compact, closed conformation of the SNARE that prevents promiscuous targeting interactions and premature insertion into membranes. Interestingly, both protein-protein and protein-lipid interactions are required for a tightly closed conformation and normal targeting.
尽管大多数SNARE蛋白的膜运输功能在从酵母到人类的进化过程中是保守的，但一些哺乳动物SNARE蛋白已经进化出了多细胞生命所特有的特殊功能。异戊二烯化的酵母SNARE蛋白Ykt6p的哺乳动物同源物可能就是这样一个例子，因为大鼠Ykt6仅在脑神经元中高度表达。此外，神经元Ykt6呈现出非常特殊的点状定位，与内膜系统的传统区室没有明显重叠，这表明Ykt6可能参与了一条对神经元功能来说独特的或经过专门修饰的途径。Ykt6靶向其独特的亚细胞位置是由其类脯肌动蛋白的长in结构域指导的。我们利用可获得的酵母Ykt6p长in结构域的高分辨率结构数据，来研究哺乳动物长in结构域控制Ykt6构象和亚细胞靶向的机制。我们发现，长in结构域的整体三级结构而非序列特异性的表面特征驱动其直接靶向Ykt6点状结构。然而，长in结构域的几个序列特异性表面特征通过分子内相互作用间接调节Ykt6定位，这些相互作用掩盖了SNARE基序和脂质基团上原本占主导地位的靶向信号。具体来说，长in结构域每一面上各有一个疏水结合口袋，以及一个混合的疏水/带电荷表面，它们参与与SNARE基序的蛋白质-蛋白质相互作用以及与分子C末端脂质基团的蛋白质-脂质相互作用。其中一个疏水口袋抑制Ykt6因蛋白质棕榈酰化而错误定位于质膜。预计Ykt6分子内相互作用会产生SNARE的紧凑、封闭构象，从而防止杂乱的靶向相互作用和过早插入膜中。有趣的是，紧密封闭的构象和正常靶向都需要蛋白质-蛋白质和蛋白质-脂质相互作用。
