Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

Abstract

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.

The Conserved ATPase Get3/Arr4 Modulates the Activity of Membrane-Associated Proteins in Saccharomyces cerevisiae

“…The npl4 mutants were initially isolated on the basis of their ability to block nuclear transport (DeHoratius and Silver 1996). Thus these RNA-processing mutants may alleviate an RNA transport defect, now presumed to be caused by the membrane defects in npl4-1 cells; alternatively, they may increase the half-life of OLE1 or npl4-1 transcripts.…”

“…The genotypes of all strains used in this study are provided in Table 1. The npl4-1 mutant strains are FY23-backcrossed strains derived from PSY825 and PSY826, which were previously described (DeHoratius and Silver 1996). Null alleles and C-terminal tags were integrated using PCR-based techniques (Baudin et al 1993;Knop et al 1999).…”

UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97

“…The Cdc48/p97 Shp1/p47 and p97 p37 complexes control the fusion of homotypic membranes [4,30], while ubiquitin-dependent protein degradation pathways require the Cdc48/p97 Ufd1-Npl4 complex [5, 14, 19, 56 -61]. The crucial role of major substrate-recruiting cofactors in these processes is reflected by the strong phenotypes of mutants in the corresponding genes in yeast: UFD1 and NPL4 are essential, and null mutants of the yeast p47 homologue SHP1 display a severe growth phenotype [7,8,59,62,63]. Surprisingly, however, shp1 mutants have also been found to exhibit defects in certain ubiquitin-dependent degradation pathways [7,8].…”

Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae

“…(Aitchison et al 1995a;Heath et al 1995), NUP1 (Bogerd et al 1994;DeHoratius and Silver 1996), NUP133 (Pemberton et al 1995), NUP85 (Goldstein et al 1996), NUP84 (Siniossoglou et al 1996), or the combination of NUP170 and POM152 (Aitchison et al 1995b) all have nuclear envelope structural abnormalities. It is possible that these previously described structural changes are related to those that facilitate SPB duplication in the absence of certain nucleoporins.…”