Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1.
SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB.
Lin
Yuan
Y
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Kimpler
Lisa A
LA
Naismith
Teresa V
TV
Lauer
Joshua M
JM
Hanson
Phyllis I
PI
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry; D001692:Biological Transport; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D010716:Phosphatidylinositols / Q000737:chemistry; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D060149:Tetraspanin 30; D014018:Tissue Distribution; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D033921:Vesicular Transport Proteins
D015703:Antigens, CD; C554586:CD63 protein, human; C481529:CHMP4A protein, human; D002352:Carrier Proteins; D003902:Detergents; D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; D010716:Phosphatidylinositols; D010980:Platelet Membrane Glycoproteins; D011108:Polymers; D012097:Repressor Proteins; D060149:Tetraspanin 30; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D012313:RNA; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C490304:VPS4B protein, human