Mechanistic insight into the nucleus-vacuole junction based on the Vac8p-Nvj1p crystal structure
Mechanistic insight into the nucleus-vacuole junction based on the Vac8p-Nvj1p crystal structure
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DOI：
10.1073/pnas.1701030114
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发表时间：
2017-06-06
影响因子：
11.1
通讯作者：
Lee, Changwook
中科院分区：
文献类型：
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作者：
Jeong, Hanbin;Park, Jumi;Lee, Changwook
核-液泡连接（NVJ）的形成是由液泡蛋白Vac8p和外核内质网膜蛋白Nvj1p直接相互作用介导的。本文报道了Vac8p与Nvj1p结合的晶体结构，分辨率为2.4埃。Vac8p包括一个灵活连接的n端H1螺旋，随后是12个犰狳重复序列（ARMs），形成右手超螺旋结构。Nvj1p延伸的80埃长环特异性结合Vac8p的ARM1-12形成的高度保守的内槽。Nvj1p-Vac8p相互作用的破坏导致紧密nvj的丧失，从而损害了酿酒酵母细胞核的片段微自噬。与Nvj1p相互作用的Vac8p阳离子三联体（Arg276， Arg317和Arg359）基序对Atg13p的识别也至关重要，Atg13p是细胞质-液泡靶向（CVT）途径的关键组成部分，表明与Vac8p的竞争性结合。事实上，阳离子三联体的突变会在体内消除Ape1p的CVT。结合生化数据，晶体结构揭示了由ARM1形成的Vac8p同型二聚体，这种自结合可能由柔性H1螺旋和Nvj1p的C端调节，对Vac8p的细胞功能至关重要。
Formation of the nucleus-vacuole junction (NVJ) is mediated by direct interaction between the vacuolar protein Vac8p and the outer nuclear endoplasmic reticulum membrane protein Nvj1p. Herein we report the crystal structure of Vac8p bound to Nvj1p at 2.4-angstrom resolution. Vac8p comprises a flexibly connected N-terminal H1 helix followed by 12 armadillo repeats (ARMs) that form a right-handed superhelical structure. The extended 80-angstrom-long loop of Nvj1p specifically binds the highly conserved inner groove formed from ARM1-12 of Vac8p. Disruption of the Nvj1p-Vac8p interaction results in the loss of tight NVJs, which impairs piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae. Vac8p cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1p are also critical to the recognition of Atg13p, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8p. Indeed, mutation of the cationic triad abolishes CVT of Ape1p in vivo. Combined with biochemical data, the crystal structure reveals a Vac8p homodimer formed from ARM1, and this self-association, likely regulated by the flexible H1 helix and the C terminus of Nvj1p, is critical for Vac8p cellular functions.