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A novel KISGQ polypeptide associated with autolysosomal membranes

一种与自体溶酶体膜相关的新型 KISGQ 多肽

基本信息

批准号：
09680629
负责人：
UENO Takashi
金额：
$ 2.11万
依托单位：
Juntendo University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680629/
关键词：
autophagy lysosome autophagosome autolysosomes proteolysis 蛋白分解オートファジ-自食作用胞

项目摘要

The membrane proteins of autolysosomes isolated from leupeptin-administered rat liver have been extensively compared with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomaal membranes were found to bemore enriched in endoplasmic reticulum lumenal proteins (protein disulfide isomerase, caireticulin, ER6O, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58K) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44k, 35k, and 32k) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and RT-PCR analyses, we identified the 44k peptide as the intact subunit of betaine homocysteine methyltransferase and the 35k and 32k peptides as two proteolytic fragments. When freshly isolated autolysosomes were incubated with pronase at 0ﾟC, both p44 and p32 were resistant to the digestion whereas p35 was completely digested. It was concluded that the 44k and 32k peptides are present in the lumen, whereas the 35k peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32k peptide occurs in the presence of E64d, showing that the 32k peptide is formed from the sequestered 44k peptide during autophagy. The accumulation is enhanced by rapamycin but completely inhibited by wortmannin, 3- methyladenine, and bafilomycin. Thus, detection of the 32k peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.

从给予亮肽素的大鼠肝脏中分离出的自溶酶体的膜蛋白已与溶酶体的膜蛋白进行了广泛的比较。除了两个膜共有的许多多肽外，还发现自体溶酶体膜更富含内质网腔蛋白（蛋白质二硫键异构酶、凯雷提库林、ER6O、BiP）和内体/高尔基体标记物（阳离子非依赖性甘露糖 6-磷酸受体、转铁蛋白受体、高尔基体） 58K）高于溶酶体膜。自体溶酶体膜蛋白包括三种多肽（44k、35k和32k），其氨基末端序列尚未报道。结合免疫印迹和 RT-PCR 分析，我们确定 44k 肽是甜菜碱同型半胱氨酸甲基转移酶的完整亚基，35k 和 32k 肽是两个蛋白水解片段。当新鲜分离的自溶酶体与链霉蛋白酶在0℃下孵育时，p44和p32都抵抗消化，而p35被完全消化。结论是 44k 和 32k 肽存在于管腔中，而 35k 肽则不存在。在原代肝细胞培养物中，饥饿诱导的 32k 肽积累发生在 E64d 存在的情况下，表明 32k 肽是在自噬过程中由隔离的 44k 肽形成的。雷帕霉素可增强积累，但渥曼青霉素、3-甲基腺嘌呤和巴弗洛霉素完全抑制积累。因此，通过免疫印迹检测 32k 肽可用作监测自噬的简化测定法。