Ubiquitin regulation of the Rab5 family GEF Vps9p.
To maintain cellular homeostasis, the levels of transmembrane receptors found on the plasma membrane must be tightly regulated. Endocytosis of activated receptors and the eventual degradation of these transmembrane proteins in the lysosome serve a vital role in maintaining the plasma membrane receptor levels as well as attenuating the downstream signaling pathways. Two processes that regulate this receptor trafficking are the covalent modification of the receptor with ubiquitin (ubiquitylation) and the activation of the Rab5 family of small GTPases. Activation of Rab5 family proteins has been shown to be critical for early steps of the endocytic pathway including delivery of activated receptors to the early endosome, while ubiquitylation of activated receptors has been shown to be involved in receptor internalization, delivery to the endosome, and sorting into the multivesiclar body. In yeast, the guanine nucleotide exchange factor Vps9p serves to integrate the activation of a Rab5 protein (Vps21p) via the Vps9 domain with ubiquitin binding via the CUE domain to facilitate the delivery of ubiquitylated receptors to the endosome. Here we provide detailed protocols for the study of Vps9p in vivo and in vitro with regard to Vps21p activation, ubiquitin binding, and Vps9p ubiquitylation.
Brian A
BA
Davies
Darren S
DS
Carney
Bruce F
BF
Horazdovsky
D001483:Base Sequence; D017931:DNA Primers; D004705:Endocytosis; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D008958:Models, Molecular; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000502:physiology; D025801:Ubiquitin / Q000502:physiology*; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*
D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D025801:Ubiquitin; D020696:rab5 GTP-Binding Proteins