Multiple roles for Rsp5p-dependent ubiquitination at the internalization step of endocytosis.
Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway. The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the alpha-factor receptor (Ste2p). Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis. Temperature-sensitive rsp5 mutant cells were defective in the internalization of alpha-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells. Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p. Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitin-dependent and -independent endocytosis signals. Constitutive Ste2p-ubiquitin internalization and fluid-phase endocytosis also required active ubiquitination machinery, including Rsp5p. These observations indicate that Rsp5p-dependent ubiquitination of a trans-acting protein component of the endocytosis machinery is required for the internalization step of endocytosis.
Dunn
R
R
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Hicke
L
L
D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000502:physiology*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000502:physiology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D043743:Ubiquitin-Protein Ligase Complexes; D014452:Ubiquitins / Q000502:physiology
D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae