The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo.
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'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and 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 combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex 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 function in the cytoplasm in a 3'-mRNA degradation pathway.
Brown
J T
JT
Section of Molecular Genetics and Microbiology and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712-1095, USA.
Bai
X
X
Johnson
A W
AW
D000998:Antiviral Agents / Q000737:chemistry* / Q000302:isolation & purification; D003593:Cytoplasm / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D000998:Antiviral Agents; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; C081929:SKI2 protein, S cerevisiae; C406671:SKI3 protein, S cerevisiae; C079882:SKI8 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins