Characterization of subcellular localization and stability of a splice variant of G alphai2

Abstract

Background: Alternative mRNA splicing of αi2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sαi2. In the sα i2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of αi2. Whereas α i2 is found predominantly at cellular plasma membranes, sαi2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sαi2 have been suggested to constitute a specific targeting signal. Results: This paper proposes and examines an alternative hypothesis: disruption of the normal Cterminus of αi2 produces an unstable protein that fails to localize to plasma membranes. sα i2 is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that αi2 and sαi2 undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sαi2 is more rapidly degraded compared to α i2. Co-expression of βγ rescues PM localization and increases expression of sαi2. In addition, αi2A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and α i2(I-331), in which the C-terminal 24 amino acids of αi2 are deleted, show a similar pattern of subcellular localization as sαi2 (i.e., intracellular membranes rather than plasma membranes). Finally, sαi2 displays a propensity to localize to potential aggresome-like structures. Conclusions: Thus, instead of the novel C-terminus of sα i2 functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of αi2 causes mislocalization and rapid degradation of sαi2. © 2002 Wedegaertner; licensee BioMed Central Ltd.