Identification of lysines within membrane-anchored Mga2p120 that are targets of Rsp5p ubiquitination and mediate mobilization of tethered Mga2p90.
Mga2p90 is an endoplasmic reticulum (ER)-localized transcription factor that is released from the ER membrane by a unique ubiquitin (Ub)-dependent mechanism. Mga2p90 mobilization requires polyubiquitination of its associating membrane-bound Mga2p120 anchor and subsequent Mga2p120-Mga2p90 complex disassembly that is mediated by ATPase Cdc48p and its heteromeric Ub-binding adaptor Npl4p-Ufd1p. Although previous studies have identified the Ub ligase (i.e., Rsp5p) and ligase-binding site on Mga2p120 that play a role in this process, the amino acids of Mga2p120 that are targets of ubiquitination and promote Mga2p90 mobilization are unknown. We have identified, using mass spectrometry analysis of in vitro ubiquitinated Mga2p120-Mga2p90 complex, that lysine residues 983 and 985 contained within the carboxy-terminal domain of Mga2p120 are Rsp5p-directed Ub-conjugation sites. Mutation of these residues as well as proximally located lysine 980 results in suppression of Mga2p120 ubiquitination in vitro and in vivo, inefficient liberation of Mga2p90 by Cdc48p(Npl4p/Ufd1p)in vitro, and ER retention of Mga2p in cells. Moreover, mga2Delta/spt23ts harboring Rsp5p binding and conjugation mga2 mutants express low OLE1 (an Mga2p90 target gene) transcripts and display reduced growth. We conclude that residues 980, 983, and 985 are targets of Rsp5p-induced polyubiquitination and mediate Cdc48p(Npl4p/Ufd1p)-dependent Mga2p90-Mga2p120 separation and Mga2p90 mobilization.
Bhattacharya
Sabyasachi
S
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Shcherbik
Natalia
N
Vasilescu
Julian
J
Smith
Jeffrey C
JC
Figeys
Daniel
D
Haines
Dale S
DS
D002853:Chromatography, Liquid; D056827:Endosomal Sorting Complexes Required for Transport; D008239:Lysine / Q000737:chemistry*; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
D056827:Endosomal Sorting Complexes Required for Transport; C542266:MGA2 protein, S cerevisiae; D008565:Membrane Proteins; D020033:Protein Isoforms; C087570:SPT23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae; D008239:Lysine