PURIFICATION AND NUCLEIC-ACID-BINDING PROPERTIES OF A SACCHAROMYCES-CEREVISIAE PROTEIN INVOLVED IN THE CONTROL OF PLOIDY

Scp160p (Saccharomyces cerevisiae protein involved in the control of p loidy), a polypeptide with a molecular mass of around 160 kDa, is asso ciated with the nuclear envelope and the endoplasmic reticulum. The mo st noteworthy phenotype of SCP160 deletion mutants is a decrease in vi ability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kuhne, C. & Karwan, A. (1995) Yeast 11, 929-944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we d emonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabd itis elegans as well as with the vigilins, vertebrate RNA-binding prot eins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) n ear its N-terminus and a potential nuclear-localization signal (NLS) b etween KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellu lose and anti-Scp160p affinity chromatography. In northwestern blottin g experiments, the electrophoretically homogeneous protein bound to ri bohomopolymers and ribosomal RNA as well as to single-stranded and dou ble-stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions an d can be released from the membrane fraction under conditions that lea d to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role fo r Scp160p in nuclear RNA export or in RNA transport within the cytopla sm.