title: Ubiquitination of the N-terminal region of caveolin-1 regulates endosomal sorting by the VCP/p97 AAA-ATPase.
abstract: Caveolin-1 (CAV1) is the defining constituent of caveolae at the plasma membrane of many mammalian cells. For turnover, CAV1 is ubiquitinated and sorted to late endosomes and lysosomes. Sorting of CAV1 requires the AAA+-type ATPase VCP and its cofactor UBXD1. However, it is unclear in which region CAV1 is ubiquitinated and how ubiquitination is linked to sorting of CAV1 by VCP-UBXD1. Here, we show through site-directed mutagenesis that ubiquitination of CAV1 occurs at any of the six lysine residues, 5, 26, 30, 39, 47, and 57, that are clustered in the N-terminal region but not at lysines in the oligomerization, intramembrane, or C-terminal domains. Mutation of Lys-5-57 to arginines prevented binding of the VCP-UBXD1 complex and, importantly, strongly reduced recruitment of VCP-UBXD1 to endocytic compartments. Moreover, the Lys-5-57Arg mutation specifically interfered with trafficking of CAV1 from early to late endosomes. Conversely and consistently, depletion of VCP or UBXD1 led to accumulation of ubiquitinated CAV1, suggesting that VCP acts downstream of ubiquitination and is required for transport of the ubiquitinated form of CAV1 to late endosomes. These results define the N-terminal region of CAV1 as the critical ubiquitin conjugation site and, together with previous data, demonstrate the significance of this ubiquitination for binding to the VCP-UBXD1 complex and for sorting into lysosomes.
authors: Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany.
authors: Philipp
authors: P
authors: Kirchner
authors: Monika
authors: M
authors: Bug
authors: Hemmo
authors: H
authors: Meyer
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D000071183:Autophagy-Related Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D021381:Protein Transport; D034622:RNA Interference; D057149:Ubiquitinated Proteins / Q000378:metabolism; D054875:Ubiquitination; D000074405:Valosin Containing Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D051242:Caveolin 1; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C533572:UBXN6 protein, human; D057149:Ubiquitinated Proteins; D001120:Arginine; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; D008239:Lysine