Yeast Nuclear Envelope Subdomains with Distinct Abilities to Resist Membrane Expansion

Abstract

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7⌬ mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7⌬ mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7⌬ mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7⌬ mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7⌬ mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA. INTRODUCTIONThe nucleus has a distinct organization, characterized by the presence of internal subcompartments. Moreover, in most, but not all, cell types the nucleus adopts a round shape. Understanding how this organization and shape are achieved is of major biological and medical interest. Certain cell types, such as those found in blood lineages (e.g., neutrophils, monocytes, and eosinophils), undergo dramatic nuclear shape changes as they differentiate (Gartner et al., 1990). Cancerous states are often associated with changes in nuclear morphology, most frequently nucleolar enlargement, changes in nuclear shape, or a combination of the two (Zink et al., 2004). Although these changes provide useful diagnostic markers of cancer progression, their mechanistic basis is still poorly understood. Other diseases are also associated with changes in nuclear shape and organization. For example, Hutchinson-Gilford progeria syndrome, which leads to premature aging, is caused by a specific mutation in the gene encoding A-type lamins and is associated with nuclear shape changes . Interestingly, progeria-like changes in nuclear shape are part of the normal aging process of nonneuronal cells in Caenorhabditis elegans (Haithcock et al., 2005). Genetic defects in the nuclear lamina are also associated with several types of muscular dystrophy . Nuclear lamins and their associated proteins provide both a rigid structure that helps shape the nuclear membrane and a platform onto which protein complexes and chromatin can bind (Holaska et al., 2002). Although much has been learned in recent years about the function of nuclear lamins, many significant questions remain. For example, it is not known how diseaseassociated changes in nuclear shape affect...

A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase

“…1B). As shown previously, in galactose-containing medium the nuclear membrane expanded in GAL-DGK1 cells (11) and similar to the nuclei of spo7Δ cells (13), expansion took place at the membrane associated with the nucleolus (Fig. 1C).…”

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

“…Immunofluorescence in Supplemental Figure S3 was performed as described previously (CAMPBELL et al 2006), except that monoclonal antibodies against alpha-tubulin (Sigma Aldrich, St. Louis, MO) were used at 1:750 dilution. Short spindles were defined as those that traversed a spherical nucleus, while long spindles traversed an hourglass nucleus with two separated DNA masses.…”

“…Short spindles were defined as those that traversed a spherical nucleus, while long spindles traversed an hourglass nucleus with two separated DNA masses. To determine nuclear morphology, cells containing a pPUS1-GFP-URA plasmid (CAMPBELL et al 2006) were grown to mid-log phase, fixed in 4% paraformaldehyde for one hour at 23˚C, and then observed using a Nikon Eclipse E800 fluorescent microscope.…”

“…In addition, the nuclear membrane is affected by altering lipid biosynthesis, for example, by inactivating the phosphatidic acid (PA) phosphohydrolase Pah1p or by inactivating the phosphates complex, made of Spo7p and Nem1p, which activates Pah1p. In the absence of Spo7p, Nem1p, or Pah1p, cells exhibit nuclear envelope extensions and extensive ER membrane sheets, and they also have altered membrane lipid composition, including a decrease in phosphatidylcholine and an increase in PA, phosphatidylethanolamine, and phosphatidylinositol (Siniossoglou et al 1998;Santos-Rosa et al 2005;Campbell et al 2006;Han et al 2006). These three proteins are unique among phospholipid biosynthesis proteins in their ability to affect nuclear morphology upon gene disruption (Han et al 2008).…”

Nucleus-Vacuole Junctions and Piecemeal Microautophagy of the Nucleus inS. cerevisiae

“…64 In Rps19p-depleted cells, aggregated preribosomal subunits were noted to partition into vacuole-associated nuclear blebs, though it remains to be determined if these blebs are bona fide PMN structures or alternate subcompartments associated with the nucleolus. 64,65 …”