Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport.
Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport.
复制标题
DOI：
10.1038/s41598-020-73276-w
复制
发表时间：
2020-10-06
影响因子：
4.6
通讯作者：
Storrie B
中科院分区：
文献类型：
--
作者：
Dickson LJ;Liu S;Storrie B
Rab 6是最丰富的高尔基体相关小GT3，由Rab 6A和Rab 6A ′两种相同的同种型组成，它们在3个氨基酸上不同，并定位于高尔基体池。这两种亚型在功能上很大程度上是冗余的，因此通常被称为Rab 6。Rab 6功能丧失抑制高尔基体逆行运输，诱导HeLa细胞中高尔基体池数量增加，并延迟顺行货物蛋白VSVG的细胞表面外观。我们假设这些影响是相互关联的，可能是由特定的脑池延迟货物运输。在脉冲追踪实验中，使用去卷积的共聚焦线扫描方法对Rab 6耗尽细胞中VSVG蛋白的tsO 45突变体的分布进行评分，我们发现在32 °C，允许条件下，通过高尔基体的顺行运输在内侧和跨高尔基池之间局部延迟，几乎是10倍。顺式向内侧转运几乎正常，反式高尔基体向TGN转运也是如此。TGN退出不受Rab 6耗尽的影响。这些效果与两种siRNA中的任一种相同。在37 °C下，使用RUSH VSVG或RUSH GPI锚定的构建体使用生物素脉冲从ER释放标记蛋白，观察到类似的高尔基体内转运延迟。使用3D-SIM，一个超分辨率的方法，我们发现，RUSH VSVG运输延迟前高尔基体。这些视觉方法表明，相对于3种不同的标记蛋白下游的反式高尔基体的顺行运输的选择性放缓。使用生物化学方法，我们发现，在Rab 6耗竭细胞中VSVG糖苷内切酶H抗性的发生被延迟。Rab 6A或Rab 6A ′异构体的单独缺失对通过高尔基体的顺行转运没有任何影响，表明Rab 6A和Rab 6A ′协同作用。延迟货物运输条件与早期电子显微镜观察到的高尔基体池的增殖密切相关。我们的研究结果强烈表明，Rab 6是选择性地需要快速顺行运输从内侧到transGolgi。我们认为，所观察到的局部脑池增殖的相关性最适合与高尔基体功能的脑池进展模型。
Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A′, that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG. We hypothesized that these effects are linked and might be explained by a cisternal-specific delay in cargo transport. In pulse chase experiments using a deconvolved, confocal line scanning approach to score the distribution of the tsO45 mutant of VSVG protein in Rab6 depleted cells, we found that anterograde transport at 32 °C, permissive conditions, through the Golgi apparatus was locally delayed, almost tenfold, between medial and trans Golgi cisterna. Cis to medial transport was nearly normal as was trans Golgi to TGN transport. TGN exit was unaffected by Rab6 depletion. These effects were the same with either of two siRNAs. Similar intra-Golgi transport delays were seen at 37 °C with RUSH VSVG or a RUSH GPI-anchored construct using a biotin pulse to release the marker proteins from the ER. Using 3D-SIM, a super resolution approach, we found that RUSH VSVG transport was delayed pre-trans Golgi. These visual approaches suggest a selective slowing of anterograde transport relative to 3 different marker proteins downstream of the trans Golgi. Using a biochemical approach, we found that the onset of VSVG endoglycosidase H resistance in Rab6 depleted cells was delayed. Depletion of neither Rab6A or Rab6A′ isoforms alone had any effect on anterograde transport through the Golgi suggesting that Rab6A and Rab6A′ act coordinately. Delayed cargo transport conditions correlate strongly with a proliferation of Golgi cisternae observed in earlier electron microscopy. Our results strongly indicate that Rab6 is selectively required for rapid anterograde transport from the medial to trans Golgi. We suggest that the observed correlation with localized cisternal proliferation fits best with a cisternal progression model of Golgi function.
登录
查看更多内容
RAB GTPases在膜交通和细胞生理学中的作用。
影响因子：
33.6
作者：
Hutagalung AH;Novick PJ
通讯作者：
Novick PJ
基于两层高尔基体的细胞器大小的控制基于内皮细胞的功能可塑性。
影响因子：
11.8
作者：
Ferraro, Francesco;Kriston-Vizi, Janos;Metcalf, Daniel J.;Martin-Martin, Belen;Freeman, Jamie;Burden, Jemima J.;Westmoreland, David;Dyer, Clare E.;Knight, Alex E.;Ketteler, Robin;Cutler, Daniel F.
通讯作者：
Cutler, Daniel F.
影响因子：
21.3
作者：
Miserey-Lenkei, S.;Chalancon, G.;Echard, A.
通讯作者：
Echard, A.
DOI：
10.1073/pnas.94.5.1828
发表时间：
1997-03-04
影响因子：
11.1
作者：
Martinez, O;Antony, C;Goud, B
通讯作者：
Goud, B
影响因子：
64.8
作者：
GOUD, B;ZAHRAOUI, A;SARASTE, J
通讯作者：
SARASTE, J