Perturbations to the ubiquitin conjugate proteome in yeast δubx mutants identify Ubx2 as a regulator of membrane lipid composition.
Perturbations to the ubiquitin conjugate proteome in yeast δubx mutants identify Ubx2 as a regulator of membrane lipid composition.
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DOI：
10.1074/mcp.m113.030163
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发表时间：
2013-10
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影响因子：
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通讯作者：
Deshaies RJ
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文献类型：
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作者：
Kolawa N;Sweredoski MJ;Graham RL;Oania R;Hess S;Deshaies RJ
酵母Cdc 48（人类细胞中的p97/VCP）是一种六聚体AAA ATP酶，被认为使用ATP水解来驱动泛素缀合蛋白与紧密结合的伴侣分离。目前的模型证实Cdc 48通过衔接蛋白与其底物连接，包括一个包含Cdc 48结合UBX结构域的7种蛋白质家族（人类中有13种）。然而，很少有特定的UBX蛋白质的底物是已知的，因此，这一假设的普遍性仍然未经检验。在这里，我们使用质谱法来鉴定在cdc 48和ubx突变体中积累的遍在蛋白缀合物。不同的Ubx突变体表现出独特的模式，共轭积累，指向个别Ubx蛋白的功能专业化。为了验证我们的研究结果，我们详细研究了内质网结合的转录因子Spt 23，我们确定为一个假定的Ubx 2底物。突变型ubx 2 Δ细胞在裂解Spt 23的泛素化120 kDa前体以形成活性p90和将p90定位于细胞核方面都有缺陷，导致编码脂肪酸去饱和酶的靶基因OLE 1的表达减少。我们的研究结果为Cdc 48的未来研究提供了资源，说明了蛋白质组学在识别特定泛素受体途径配体方面的实用性，并揭示了Ubx 2在膜脂质生物合成调控中的关键作用。
Yeast Cdc48 (p97/VCP in human cells) is a hexameric AAA ATPase that is thought to use ATP hydrolysis to power the segregation of ubiquitin-conjugated proteins from tightly bound partners. Current models posit that Cdc48 is linked to its substrates through adaptor proteins, including a family of seven proteins (13 in human) that contain a Cdc48-binding UBX domain. However, few substrates for specific UBX proteins are known, and hence the generality of this hypothesis remains untested. Here, we use mass spectrometry to identify ubiquitin conjugates that accumulate in cdc48 and ubx mutants. Different ubx mutants exhibit unique patterns of conjugate accumulation that point to functional specialization of individual Ubx proteins. To validate our findings, we examined in detail the endoplasmic reticulum-bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. Mutant ubx2Δ cells are deficient in both cleaving the ubiquitinated 120 kDa precursor of Spt23 to form active p90 and in localizing p90 to the nucleus, resulting in reduced expression of the target gene OLE1, which encodes fatty acid desaturase. Our findings provide a resource for future investigations on Cdc48, illustrate the utility of proteomics to identify ligands for specific ubiquitin receptor pathways, and uncover Ubx2 as a key player in the regulation of membrane lipid biosynthesis.