title: Distinct subclasses of small GTPases interact with guanine nucleotide exchange factors in a similar manner.
abstract: The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3-loop 7 [alpha3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and alpha3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and alpha3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.
authors.affiliation: University of Southern California/Norris Cancer Center and Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Day
authors.forename: R D
authors.initials: RD
authors.lastname: Mosteller
authors.forename: D
authors.initials: D
authors.lastname: Broek
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D013489:Suppression, Genetic / Q000235:genetics; D020691:rab GTP-Binding Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C079548:DSS4 protein, S cerevisiae; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D018631:ras Proteins