Li Xia, Liu Dongmei, Griffis Eric, Novick Peter
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, United States.
Nikon Imaging Center, University of California at San Diego, La Jolla, California, United States.
bioRxiv. 2023 Feb 10:2023.02.09.527811. doi: 10.1101/2023.02.09.527811.
Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport because anterograde vesicles are marked with a different Rab than retrograde vesicles. To explore this proposal, we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab, Ypt51. The Sec2GEF-GFP-CUE construct was found to localize to bright puncta predominantly near sites of polarized growth and this localization was strongly dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. The puncta appeared to consist of clusters of 80 nm vesicles and although the puncta are largely static, FRAP analysis suggests that traffic into and out of these clusters continues. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth, where they tethered and fused with the plasma membrane despite the misdirection of Sec4 and its effectors. In total, the results suggest that while Rabs play a critical role in regulating vesicular transport, cells are remarkably tolerant of Rab misdirection.
双向囊泡运输连接了沿胞吐和胞吞途径的各个区室。Rab GTP酶参与确定囊泡运输的方向，因为顺行囊泡与逆行囊泡标记的Rab不同。为了探究这一观点，我们试图通过将Sec4鸟苷酸交换因子（GEF）Sec2的催化结构域融合到Vps9的CUE定位结构域上，将胞吐Rab Sec4重定向到胞吞囊泡上，Vps9是胞吞Rab Ypt51的GEF。发现Sec2GEF-GFP-CUE构建体主要定位于极化生长位点附近的明亮斑点，并且这种定位强烈依赖于CUE结构域与添加到注定要进行胞吞内化的蛋白质细胞质尾巴上的泛素部分结合的能力。Sec4及其效应器以不同效率被招募到这些斑点上。这些斑点似乎由80 nm的囊泡簇组成，尽管这些斑点在很大程度上是静止的，但荧光恢复后光漂白（FRAP）分析表明进出这些簇的运输仍在继续。表达Sec2GEF-GFP-CUE的细胞生长得非常好，并且以接近正常的效率分泌蛋白质，这意味着高尔基体衍生的分泌囊泡被递送到细胞生长的极化位点，尽管Sec4及其效应器的定向错误，但它们在那里拴系并与质膜融合。总的来说，结果表明虽然Rab在调节囊泡运输中起关键作用，但细胞对Rab的定向错误具有显著的耐受性。
