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.