The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex invivo

The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-e xoribonuclease responsible for the majority of cytoplasmic RNA turnover, wh ereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA a nd for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combinat ion with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, w e show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an appar ent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 st oichiometry of the proteins in the complex. Ski2p did not associate with Sk i3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the abse nce of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. I n addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect c omplex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p f unction in the cytoplasm in a 3'-mRNA degradation pathway.