STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes.
STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway.
Bache
Kristi G
KG
Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
Raiborg
Camilla
C
Mehlum
Anja
A
Stenmark
Harald
H
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000172:deficiency; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D002966:Clathrin; C486206:EPS15 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; C109775:STAM protein, human; C491647:STAM2 protein, human; D029701:Saccharomyces cerevisiae Proteins; C491648:Stam protein, mouse; C491646:Stam2 protein, mouse; D025801:Ubiquitin; C119627:VPS27 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D066246:ErbB Receptors; D000251:Adenosine Triphosphatases