Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.
Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.
From the Department of Biochemistry and Biophysics.
Gauri
G
Dixit
From the Department of Biochemistry and Biophysics.
Rachael
R
Baker
Department of Biology, and.
Carly M
CM
Sacks
School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332.
Matthew P
MP
Torres
From the Department of Biochemistry and Biophysics, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 and hdohlman@med.unc.edu.
Henrik G
HG
Dohlman
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003433:Crosses, Genetic; D004705:Endocytosis; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009024:Morphogenesis; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D059008:Time-Lapse Imaging; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
C099939:BUL1 protein, S cerevisiae; D002352:Carrier Proteins; C108125:DDI1 protein, S cerevisiae; C510360:Ede1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
Endocytosis; G Proteins; Protein Domains; Protein Purification; Ubiquitination