ABSTRACT
The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (approximately 350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.
Subject(s)
Carrier Proteins/physiology , DNA-Binding Proteins/physiology , Endocytosis/physiology , Endosomes/metabolism , Fungal Proteins/physiology , Saccharomyces cerevisiae Proteins , Transcription Factors/physiology , Vesicular Transport Proteins , 3T3 Cells/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cathepsin D/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endosomal Sorting Complexes Required for Transport , ErbB Receptors/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Lysosomes/metabolism , MAP Kinase Signaling System , Macromolecular Substances , Mice , Molecular Sequence Data , Protein Transport , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, IGF Type 2/metabolism , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.
