Importance of the N-terminal domain of the Qb-SNARE Vti1p for different membrane transport steps in the yeast endosomal system.
Importance of the N-terminal domain of the Qb-SNARE Vti1p for different membrane transport steps in the yeast endosomal system.
复制标题
QB-SNARE VTI1P的N末端结构域对于酵母内体系统中不同膜传输步骤的重要性。
DOI：
10.1371/journal.pone.0066304
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Fischer von Mollard G
中科院分区：
文献类型：
--
作者：
Gossing M;Chidambaram S;Fischer von Mollard G
运输囊泡和靶膜上的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）对于囊泡靶向和融合至关重要。它们形成SNARE复合物，其中包含由三个或四个不同SNARE贡献的四个α-螺旋SNARE基序。大多数SNARE仅在单个传输步骤中起作用。酵母SNARE Vti 1 p参与四个不同的SNARE复合物的运输从trans-Golgi网络到晚期内体，在运输到液泡，在逆行运输从内体到trans-Golgi网络和在逆行运输内的高尔基体。到目前为止，所有研究的vti 1突变体都在SNARE基序内发生突变。Vti 1 p的N-末端结构域形成一个三螺旋束，称为Habc结构域，其功能知之甚少。在这里，我们产生了一个温度敏感的突变体的这个域，研究不同的运输步骤的影响。用圆二色谱分析野生型和vti 1 -3 Habc结构域的二级结构。在温度敏感的vti 1 -3突变体中鉴定的氨基酸交换引起Habc结构域的展开。运输途径进行了研究，通过免疫沉淀新合成的蛋白质后，脉冲追踪标记和荧光显微镜的GFP标记的蛋白质之间的质膜，早期内体和高尔基体循环。在vti 1 -3细胞中，在37°C下，晚期内体的转运和晚期内体陷阱复合物的组装被阻断。逆行运输到transGolgi网络的影响，而与液泡融合是可能的，但速度较慢。介导这些步骤的SNARE复合物的稳态水平比晚期内体SNARE复合物的稳态水平受影响较小。由于不同的运输步骤受到影响，我们的数据表明，一个折叠的Vti 1 p Habc域运输的重要性。
SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) on transport vesicles and target membranes are crucial for vesicle targeting and fusion. They form SNARE complexes, which contain four α-helical SNARE motifs contributed by three or four different SNAREs. Most SNAREs function only in a single transport step. The yeast SNARE Vti1p participates in four distinct SNARE complexes in transport from the trans Golgi network to late endosomes, in transport to the vacuole, in retrograde transport from endosomes to the trans Golgi network and in retrograde transport within the Golgi. So far, all vti1 mutants investigated had mutations within the SNARE motif. Little is known about the function of the N-terminal domain of Vti1p, which forms a three helix bundle called Habc domain. Here we generated a temperature-sensitive mutant of this domain to study the effects on different transport steps. The secondary structure of wild type and vti1-3 Habc domain was analyzed by circular dichroism spectroscopy. The amino acid exchanges identified in the temperature-sensitive vti1-3 mutant caused unfolding of the Habc domain. Transport pathways were investigated by immunoprecipitation of newly synthesized proteins after pulse-chase labeling and by fluorescence microscopy of a GFP-tagged protein cycling between plasma membrane, early endosomes and Golgi. In vti1-3 cells transport to the late endosome and assembly of the late endosomal SNARE complex was blocked at 37°C. Retrograde transport to the trans Golgi network was affected while fusion with the vacuole was possible but slower. Steady state levels of SNARE complexes mediating these steps were less affected than that of the late endosomal SNARE complex. As different transport steps were affected our data demonstrate the importance of a folded Vti1p Habc domain for transport.
登录
查看更多内容
影响因子：
11.4
作者：
Holthuis, JCM;Nichols, BJ;Pelham, HRB
通讯作者：
Pelham, HRB
影响因子：
4.5
作者：
Fridmann-Sirkis, Y;Kent, HM;Pelham, HRB
通讯作者：
Pelham, HRB
影响因子：
3.3
作者：
Burd, CG;Peterson, M;Emr, SD
通讯作者：
Emr, SD
影响因子：
4
作者：
Chidambaram, Subbulakshmi;Zimmermann, Jana;von Mollard, Gabriele Fischer
通讯作者：
von Mollard, Gabriele Fischer
DOI：
10.1038/85012
发表时间：
2001-03-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Dulubova, I;Yamaguchi, T;Rizo, J
通讯作者：
Rizo, J