细胞自噬是一种严密调控的降解和回收蛋白质和细胞器的分解代谢过程。泛素化在细胞自噬调控中起重要作用。液泡膜蛋白1（Vacuole Membrane Protein 1，VMP1）是一种必需的自噬蛋白质。在携带激活的Kirsten大鼠肉瘤病毒肿瘤基因同源物（KRAS）的胰腺癌干细胞中，VMP1的表达引发细胞自噬并使其对治疗产生抗性。通过生物化学和细胞学方法，我们确定了泛素化作为VMP1蛋白在自噬体生物合成的初始阶段的一种翻译后修饰。VMP1作为自噬体膜的一部分保持泛素化状态，直到自溶体形成，自噬通量期间。然而，VMP1既不被自噬降解，也不被泛素-蛋白酶体系统降解。质谱和免疫沉淀显示细胞分裂周期蛋白Cdt2是VMP1的一个新的相互作用蛋白，并参与其中。基于CRL（cullin-RING ubiquitin ligase complex 4）蛋白质体内酶复合物相关癌相关酶的基质识别亚基Cdt2，VMP1的泛素化在这之下减少，CRL抑制剂MLN4924下则增加。此外，CRL抑制显著影响VMP1招募和自噬体形成。我们的结果表明，在人类肿瘤细胞中，泛素化是VMP1在自噬期间的一种新的翻译后修饰。VMP1的泛素化可能与肿瘤细胞对治疗的抗性具有相关临床意义。

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin-RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance.