Characterization of subcellular localization and stability of a splice variant of G alpha(i2)

BACKGROUND: Alternative mRNA splicing of α(i2), a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sα(i2). In the sα(i2) splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of α(i2). Whereas α(i2) is found predominantly at cellular plasma membranes, sα(i2) has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sα(i2) have been suggested to constitute a specific targeting signal. RESULTS: This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of α(i2) 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 α(i2) and sα(i2) undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sα(i2) is more rapidly degraded compared to α(i2). Co-expression of βγ rescues PM localization and increases expression of sα(i2). In addition, α(i2)A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and α(i2)(1–331), in which the C-terminal 24 amino acids of α(i2) are deleted, show a similar pattern of subcellular localization as sα(i2) (i.e., intracellular membranes rather than plasma membranes). Finally, sα(i2) 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 α(i2) causes mislocalization and rapid degradation of sα(i2).