G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase.
Emerging evidence suggests that ubiquitination serves as a protein trafficking signal in addition to its well characterized role in promoting protein degradation. The yeast G protein alpha subunit Gpa1 represents a rare example of a protein that undergoes both mono- and poly-ubiquitination. Whereas mono-ubiquitinated Gpa1 is targeted to the vacuole, poly-ubiquitinated Gpa1 is directed instead to the proteasome. Here we investigate the structural requirements for mono- and poly-ubiquitination of Gpa1. We find that variants of Gpa1 engineered to be unstable are more likely to be poly-ubiquitinated and less likely to be mono-ubiquitinated. In addition, mutants that cannot be myristoylated are no longer mono-ubiquitinated but are still polyubiquitinated. Finally, we show that the ubiquitin ligase Rsp5 is necessary for Gpa1 mono-ubiquitination in vivo and that the purified enzyme is sufficient to catalyze Gpa1 mono-ubiquitination in vitro. Taken together, these data indicate that mono- and poly-ubiquitination have distinct enzyme and substrate recognition requirements; whereas poly-ubiquitination targets misfolded protein for degradation, a distinct ubiquitination apparatus targets the fully mature, fully myristoylated G protein for mono-ubiquitination and delivery to the vacuole.
Torres
Matthew P
MP
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Lee
Michael J
MJ
Ding
Feng
F
Purbeck
Carrie
C
Kuhlman
Brian
B
Dokholyan
Nikolay V
NV
Dohlman
Henrik G
HG
D056827:Endosomal Sorting Complexes Required for Transport; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*; D014617:Vacuoles / Q000201:enzymology* / Q000235:genetics
D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C469287:RSP5 protein, S cerevisiae