ABSTRACT
The microsomal fraction isolated form serine palmitoyltransferase (lcb2/scs1) mutants is enriched in a 90 kDa protein. The protein was identified as the major coat (Gag) protein of the L-A dsRNA virus particles by partial sequencing and by its interaction with anti-Gag antibodies. The total amount of Gag in whole-cell lysates of scs1/lcb2 mutant cells is greater than in wild-type lysates indicating that the enrichment of the protein in the microsomal fraction of scs1/lcb2 mutant cells may result from increased copy number of the L-A dsRNA virus. This is supported by the findings that the mutants also have increased levels of L-A dsRNA. Altered sphingolipid synthesis in the scs1 mutant cells appears to increase the copy number of the L-A viral particles.
Subject(s)
Acyltransferases/genetics , RNA Viruses/isolation & purification , RNA, Double-Stranded/isolation & purification , RNA, Viral/isolation & purification , Saccharomyces cerevisiae/virology , Amino Acid Sequence , Antibodies, Viral , Capsid/analysis , Capsid/chemistry , Capsid/genetics , Gene Products, gag/analysis , Gene Products, gag/chemistry , Gene Products, gag/genetics , Microsomes/virology , Molecular Sequence Data , Molecular Weight , Mutation/physiology , Peptide Fragments/chemistry , RNA Viruses/immunology , Recombinant Fusion Proteins , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins , Sequence Analysis , Serine C-Palmitoyltransferase , Sphingolipids/biosynthesisABSTRACT
A previous report from this laboratory described an estrogen-regulated endoribonuclease activity on Xenopus liver polysomes which had properties one might expect for a messenger ribonuclease involved in the regulated destabilization of albumin mRNA (Pastori, R. L., Moskaitis, J. E., and Schoenberg, D. R. (1991) Biochemistry 30, 10490-10498). This report describes the purification and properties of this ribonuclease. The purified nuclease fraction contained a doublet of 62 and 64 kDa and a small amount of a 40-kDa peptide. In situ analysis on both denaturing and nondenaturing gels using an albumin transcript as substrate showed all three proteins possess nuclease activity. Peptide mapping and Western blot with a polyclonal antiserum showed the 62- and 64-kDa peptides to be isoforms, and the 40-kDa peptide to be a degradation product of the larger species. Two-dimensional gel electrophoresis further separated the 62- and 64-kDa species into three pairs of proteins, with isoelectric points of 9.6, 9.8, and 9.8. The purified ribonuclease rapidly degraded a full-length albumin transcript, yet had no effect on either a full-length albumin antisense transcript or full-length ferritin transcript. A number of properties of the purified nuclease were characterized, including the effects of salt, divalent cations, EDTA, sulfhydryl reagents, and temperature. Treatment of the polysomal nuclease with micrococcal nuclease had no effect, indicating that this enzyme does not require an RNA cofactor for activity. Finally, primer extension mapped the major cleavage site to an overlapping repeated sequence APyrUGA, with cleavage between and adjacent to the two pyrimidine residues generating fragments with 5'-hydroxyls.
