The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells.
We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.
Ward
Diane McVey
DM
University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, Utah 84132, USA.
Vaughn
Michael B
MB
Shiflett
Shelly L
SL
White
Paul L
PL
Pollock
Amanda L
AL
Hill
Joshua
J
Schnegelberger
Rachel
R
Sundquist
Wesley I
WI
Kaplan
Jerry
J
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism / Q000821:virology*; D000939:Epitopes / Q000737:chemistry; D005347:Fibroblasts / Q000821:virology; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
C499838:CHMP5 protein, human; D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D000939:Epitopes; D034741:RNA, Small Interfering; C499837:VTA1 protein, human; D049452:Green Fluorescent Proteins