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J. Biol. Chem., Vol. 281, Issue 43, 32596-32605, October 27, 2006

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Gib2, A Novel G-like/RACK1 Homolog, Functions as a G Subunit in cAMP Signaling and Is Essential in Cryptococcus neoformans^*

Daniel A. Palmer¹, Jill K. Thompson¹, Lie Li, Ashton Prat, and Ping Wang^¶2

From the Research Institute for Children, Departments of Pediatrics and ^¶Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70118

Canonical G proteins are heterotrimeric, consisting of , , and subunits. Despite multiple G subunits functioning in fungi, only a single G subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the G subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel G-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed propeller structure characteristic of transducins. Gib2 is also shown to interact, respectively, with two G subunit homologs, Gpg1 and Gpg2, similar to the conventional G subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.

Received for publication, March 23, 2006 , and in revised form, August 24, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY954369 [GenBank] , AY954370 [GenBank] , AY907677 [GenBank] , AY907678 [GenBank] , AY907679 [GenBank] , and AY907680 [GenBank] .

^* This work was supported by National Institutes of Health Grant AI054958 and a fund from the Children's Hospital of New Orleans, LA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: 200 Henry Clay Ave., New Orleans, LA 70118. Tel.: 504-896-2739; Fax: 504-894-5379; E-mail: pwang{at}lsuhsc.edu.

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