ROLE OF NOVEL KELCH REPEAT PROTEINS IN YEAST DIFFERENTIATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Heitman lab studies a novel G protein receptor that allows budding yeast cells to sense glucose and undergo pseudohyphal filamentous growth. The receptor is coupled to a G protein alpha subunit, Gpa2, which has all the hallmarks of a heterotrimeric Ga subunit but does not function with the known yeast Gbg subunits Ste4 and Ste18. Our studies revealed two novel homologous proteins, Gpb1 and Gpb2, function as Gb structural mimics to inhibit signaling by Gpa2 and an unknown target in the signaling pathway. Most interestingly, the Gpb1 and Gpb2 proteins lack the 7 signature WD-40 motifs characteristic of b subunits. Instead, both have 7 kelch repeats, previously implicated in protein-protein interactions. The structure of an enzyme containing 7 kelch repeats, galactose oxidase, is known. This protein adopts a 7-bladed b-propeller structure essentially superimposable upon the known X-ray crystal structures of Gbg. These studies reveal a novel class of heterotrimeric G proteins, and related b subunit mimics may function in multicellular eukaryotes. One of us has FLAG-tagged Gpb1 and Gpb2. Gpb1-FLAG (C-terminally tagged construct) or FLAG-Gpb2 (N-terminally tagged construct) were expressed from an attenuated ADH1 promoter on a 2 um plasmid in a gpb1,2 gpa2 triple mutant yeast strain background. Interacting proteins were eluted by adding FLAG peptide. On both silver staining and Coomassie staining, specific bands were apparent. Current work is to FLAG-tag both the receptor and the coupled G alpha subunit Gpa2. We anticipate that these studies will reveal novel subunits (such as the gamma subunit, for which we have a strong candidate) as well as the relevant regulatory proteins and effectors. Since there is quite a bit known about the Ras-cAMP signaling pathway in yeast, there are many candidates, including Ras1/2, Cap1, adenylyl cylase, Ira1/2, Pde1,2, and the subunits of protein kinase A, which should make interpretation of the mass spectrometry interaction data straightforward.