A single PXY motif located within the carboxyl terminus of Spt23p and Mga2p mediates a physical and functional interaction with ubiquitin ligase Rsp5p.
Proteasome-dependent processing of the endoplasmic reticulum localized transcription factor Spt23p of Saccharomyces cerevisiae generates its transcriptionally competent form and requires the WW domain containing Rsp5p ubiquitin ligase. Although previous studies documented an Rsp5p-Spt23p association in cells, very little is known about the nature of this interaction. We report here the identification of an imperfect type I WW domain-binding site (LPKY) within the carboxyl-terminal region of Spt23p that is required for Rsp5p binding in vitro and in vivo. Deletion of this motif abrogates Rsp5p-induced ubiquitination of Spt23p in vitro and reduces ubiquitination of the Spt23p precursor in yeast. In addition, the Spt23pDeltaLPKY mutant is inefficiently processed and is defective at up-regulating target gene (OLE1) expression in cells. Deletion of the corresponding LPKY site within Mga2p, an Spt23p homologue, also abrogates Rsp5p binding and Rsp5p-dependent ubiquitination in vitro as well as Rsp5p binding and Mga2p polyubiquitination in cells. However, the Mga2pDeltaLPKY mutant undergoes efficient proteasome-dependent processing. These experiments indicate that the LPKY motif of Spt23p is required for Rsp5p binding, Rsp5-induced ubiquitination, proteasome-dependent processing, and its OLE1 inducing function. They also suggest that the LPKY motif of Mga2p is required for Rsp5p binding and ubiquitination, and Rsp5p regulates Mga2p function by a mechanism that is independent of providing the partial degradation signal.
Shcherbik
Natalia
N
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad St., Philadelphia, PA 19140, USA.
Kee
Younghoon
Y
Lyon
Nancy
N
Huibregtse
Jon M
JM
Haines
Dale S
DS
D020816:Amino Acid Motifs; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D056827:Endosomal Sorting Complexes Required for Transport; D017353:Gene Deletion; D047468:Immunoprecipitation; D008565:Membrane Proteins; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; C087570:SPT23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; C469287:RSP5 protein, S cerevisiae