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A novel complex of membrane proteins required for formation of a spherical nucleus

1998; Springer Nature; Volume: 17; Issue: 22 Linguagem: Inglês

10.1093/emboj/17.22.6449

1460-2075

Symeon Siniossoglou,

RNA and protein synthesis mechanisms

Article16 November 1998free access A novel complex of membrane proteins required for formation of a spherical nucleus Symeon Siniossoglou Symeon Siniossoglou BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany S.Siniossoglou and H.Santos-Rosa contributed equally to this work Search for more papers by this author Helena Santos-Rosa Helena Santos-Rosa BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany S.Siniossoglou and H.Santos-Rosa contributed equally to this work Search for more papers by this author Juri Rappsilber Juri Rappsilber EMBL, Meyerhofstrasse1, D-69117 Heidelberg, Germany Search for more papers by this author Mathias Mann Mathias Mann Center for Experimental Bioinformatics, Odense University, Odense, Denmark Search for more papers by this author Ed Hurt Corresponding Author Ed Hurt BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Symeon Siniossoglou Symeon Siniossoglou BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany S.Siniossoglou and H.Santos-Rosa contributed equally to this work Search for more papers by this author Helena Santos-Rosa Helena Santos-Rosa BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany S.Siniossoglou and H.Santos-Rosa contributed equally to this work Search for more papers by this author Juri Rappsilber Juri Rappsilber EMBL, Meyerhofstrasse1, D-69117 Heidelberg, Germany Search for more papers by this author Mathias Mann Mathias Mann Center for Experimental Bioinformatics, Odense University, Odense, Denmark Search for more papers by this author Ed Hurt Corresponding Author Ed Hurt BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Symeon Siniossoglou1,4, Helena Santos-Rosa1,4, Juri Rappsilber2, Mathias Mann3 and Ed Hurt 1 1BZH, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany 2EMBL, Meyerhofstrasse1, D-69117 Heidelberg, Germany 3Center for Experimental Bioinformatics, Odense University, Odense, Denmark 4S.Siniossoglou and H.Santos-Rosa contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6449-6464https://doi.org/10.1093/emboj/17.22.6449 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast. One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution. Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins. Nem1p binds to Spo7p via its conserved C-terminal domain. Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, pore-containing double nuclear membrane extensions. These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm. Interestingly, not only Spo7− and Nem1−, but also several nucleoporin mutants are defective in sporulation. Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division. Introduction In eukaryotic cells, the nuclear envelope separates the nuclear from the cytoplasmic compartment. Nuclear pore complexes (NPCs) which are embedded in the nuclear membrane regulate transport between these two compartments (for a review see Panté and Aebi, 1995; Doye and Hurt, 1997). Based on electron microscopy studies, the NPC has been proposed to consist of a central spoke assembly with 8-fold symmetry to which a cytoplasmic and nuclear ring are attached. From these rings, filaments emanate towards the cytoplasm and nucleus, respectively. Although the yeast NPC is slightly smaller than its vertebrate counterpart, the overall architecture of the NPC is conserved in all eukaryotic cells (Rout and Blobel, 1993; Yang et al., 1998). During the past years, many NPC-associated proteins (nucleoporins) have been identified and characterized, particularly in the yeast Saccharomyces cerevisiae, where ∼30 nucleoporins are known to date, and many of them are organized in distinct NPC subcomplexes (for a review, see Doye and Hurt, 1997). In contrast to the increasing knowledge of the composition of NPCs, very little is known about how and where this complex organelle assembles. It is believed that a fusion event between the outer and inner nuclear membrane generates a hole in the envelope, which is then followed by the ordered assembly of individual nucleoporins or their respective subcomplexes. Indeed, intermediates during NPC biogenesis recently have been visualized in growing nuclear envelopes from Xenopus egg extracts (Goldberg et al., 1997). However, the role of transmembrane proteins in the biogenesis and assembly of the NPCs is not clear, since deletion of the only known integral pore membrane protein in yeast, Pom152p, does not affect cell growth (Wozniak et al., 1994). Thus, other components of the nuclear envelope must participate in the fusion and anchoring of NPCs within the nuclear pore membrane. It has been suggested that Ndc1p, an essential membrane protein required for the insertion of the spindle pole body (SPB) into the nuclear envelope, might play a similar role during NPC insertion and assembly (Winey et al., 1993). The nuclear envelope is composed of two distinct membranes enclosing the perinuclear space (for a review, see Goldberg and Allen, 1995; Gant and Wilson, 1997). The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and thus also functions in protein translocation and secretion. The inner nuclear membrane in higher eukaryotes is covered by the nuclear lamina, which faces the nucleoplasm and participates in the organization of the chromatin and nucleoskeleton. However, the yeast nuclear envelope, which in contrast to higher eukaryotes does not break down during nuclear division, appears not to have an underlying nuclear lamina. During interphase, the surface of the nuclear envelope in yeast cells increases (Jordan et al., 1977; Winey et al., 1997), but the mechanism of nuclear membrane biogenesis and how this is coordinated with NPC assembly is not known. There are several examples where the biogenesis and organization of the nuclear membrane in yeast is impaired; in one case, overexpression of membrane proteins such as HMG-CoA reductase isoform 1 (HMG1) (Wright et al., 1988), cytochrome b5 (Vergeres et al., 1993) or chicken lamin B receptor (Smith and Blobel, 1994) caused a striking proliferation of the nuclear membrane, forming a stack of paired membranes which are arranged in layers around the nucleus. This structure, which was called ‘karmellae’ and corresponds to a proliferated ER membrane, is free of NPCs. The link between the overproduction of a membrane protein and the generation of karmellae is not clear, although it was reported that a luminal domain of HMG1 is responsible for the membrane proliferation (Parrish et al., 1995). Drastic nuclear membrane perturbations, which are completely different from ‘karmellae’ membrane stacks, have been observed in nucleoporin mutants. The structural abnormalities of the nuclear envelope concern both NPC and nuclear membrane organization. These include grape-like arrangements of both NPCs and nuclear membrane domains, for instance in nup145 mutants (Wente and Blobel, 1994), an outer nuclear membrane seal over the NPCs, as in the case of nup116 mutants (Wente and Blobel, 1993), or cells with long nuclear envelope projections, as in nup1 mutants (Bogerd et al., 1994) or nup170pom152 double mutants (Aitchison et al., 1995). Drastic structural defects of the nuclear envelope were also observed in nup85 mutants, and to a lesser extent in nup84-disrupted cells (Goldstein et al., 1996; Siniossoglou et al., 1996). In particular, the nuclear envelope of nup85-disrupted cells has protrusions which come in contact with each other, thereby engulfing part of the cytoplasmic compartment. At these contact zones, the nuclear pores clustered extensively, forming grape- or blister-like NPC/nuclear membrane arrangements. Mutations in MTR7/ACC1, which encodes acetyl-CoA-carboxylase, a key enzyme in fatty acid biosynthesis, also cause an expansion of the perinuclear space due to the wide separation of outer and inner nuclear membrane, and protuberances extending from the inner membrane into the intermembrane space (Schneiter et al., 1996). This was taken as evidence for a specific need for lipids with very long fatty acid chains to stabilize the insertion of NPCs at the pore-membrane interface and allow for a correct nuclear membrane biogenesis. Finally, the integral nuclear/ER membrane protein Snl1p was found recently in a genetic screen for high copy suppressors of a temperature-sensitive mutant overproducing the Nup116p C-terminal domain (Ho et al., 1998). It was suggested that Snl1p plays a role in stabilizing NPC structure and function because its overproduction can rescue the nuclear membrane herniations of nup116 mutants. We previously have identified the Nup84p nucleoporin complex consisting of six subunits, with an essential role in nuclear envelope/NPC organization and mRNA export (Siniossoglou et al., 1996). Strikingly, this complex contains Sec13p, a COPII coat protein involved in vesicular transport from the ER to the Golgi (Pryer et al., 1993). This suggested a link between distinct steps in nuclear membrane and pore biogenesis, and vesicular transport. Furthermore, the Nup84p complex plays a crucial role in correct NPC distribution within the nuclear membrane (see above). In this study, we extend these studies of nuclear envelope organization in yeast and describe a novel complex consisting of two nuclear/ER membrane proteins, Spo7p and Nem1p, which were found in a genetic screen with a nup84 disruption allele. Both Spo7p and Nem1p are essential for the maintenance of normal spherical nuclear morphology, but do not participate in nucleocytoplasmic transport reactions. Surprisingly, we found that not only the Spo7p-Nem1p complex, but also a subset of nucleoporins, including Nup84p, are essential for sporulation, suggesting a so far unrecognized link between this process and nuclear envelope/NPC organization. Results A protein required for sporulation and a novel conserved protein genetically interact with the nucleoporin Nup84p Nup84p is a member of a large nucleoporin complex (Siniossoglou et al., 1996). Yeast cells lacking NUP84 show a moderate defect in mRNA export as compared with nup85, nup120 and nup145 mutants, but are strongly impaired in nuclear pore distribution. This prompted us to use the nup84 disruption strain in a synthetic lethal screen in order to isolate novel factors involved in nuclear membrane and pore biogenesis (see Materials and methods). Among the 25 000 mutagenized colonies, nine mutants were synthetically lethal (sl) with the nup84::HIS3 disruption allele. The complementing genes of five of these sl mutants were identified. Sl243 and sl384 were complemented by the nucleoporin gene NSP1 (Hurt, 1988) and sl273 by another nucleoporin gene, NUP116 (Wente et al., 1992; Wimmer et al., 1992). Sl269 was complemented by SPO7 (DDBJ/EMBL/GenBank accession No. 349744; SGD accession No. YAL009w) and sl235 by a novel gene (DDBJ/EMBL/GenBank accession No. 500822; SGD accession No. YHR004c) designated NEM1 (for nuclear envelope morphology; see also below) (Figure 1A). NEM1 encodes a protein of 446 amino acids with a predicted mol. wt of 50 kDa. SPO7 encodes a protein of 30 kDa and was identified many years ago as a gene required for sporulation in yeast (Esposito and Esposito, 1969, 1974). Figure 1.SPO7 and NEM1 are genetically linked to NUP84. (A) Cloning of SPO7 and NEM1 by complementation of two synthetically lethal mutants derived from the nup84::HIS3 sl screen. Sl mutants sl269 and sl235 were transformed with the indicated pUN100-LEU2 plasmids containing no insert, or NUP84, SPO7 and NEM1, respectively. Transformants were grown at 30°C on 5-FOA-containing plates for 3 days. Growth on this plate indicates that the synthetically lethal phenotype of the sl mutant was complemented by the corresponding genes. (B) Growth properties of Spo7− and Nem1− strains. Pre-cultures of spo7::HIS3 and nem1::HIS3 strains (Spo7−, Nem1−), as well as the same strains complemented by the wild-type SPO7 and NEM1 genes (Spo7+, Nem1+) present on a ARS/CEN plasmid, respectively, were diluted in liquid YPD medium, and an equivalent number of cells (undiluted, 1/10 and 1/100 diluted) were spotted onto YPD plates and incubated at 30 or 37°C for 3 days. (C) Multiple sequence alignment of the Nem1p C-terminal domain (residues 233-446) with related ORFs found in the data libraries. Two human genes, HYA22 (H.s.1; DDBJ/EMBL/GenBank accession No. D88153, residues 154-340) and OS4 (H.s.2; accession No. AF000152, residues 81-283), two C.elegans ORFs (C.e.1; accession No. B0379.4, residues 50-288 and C.e.2; accession No. F45E12.1, residues 37-246), one S.pombe ORF (S.p.1; Swissprot accession No. Q09695, residues 144-325) and two S.cerevisiae ORFs (S.c.1; SGD accession No. YLL010c, residues 237-427 and S.c.2; SGD accession No. YLR019w, residues 207-397) were aligned using ClustalW1.7 and displayed with the program ‘Boxshade’ (http://ulrec3.unil.ch/software/box_form.html). Download figure Download PowerPoint Although Spo7p and Nem1p are not homologous, they share some common features in their protein sequences. Both are very basic (isoelectric point of 10.1 for Spo7p and of 9.1 for Nem1p) and contain in their primary sequence extended stretches of hydrophobic amino acids, suggesting that they might be membrane proteins (see Figure 3A). A database search revealed no Spo7p homologues in other organisms so far. However, several open reading frames (ORFs) in different organisms, including Schizosaccharomyces pombe, Caenorhabditis elegans and human, exhibit high homology within the 200 C-terminal amino acids of Nem1p (Figure 1C). Furthermore, three additional uncharacterized ORFs (YLR019w, YLL010c and YPL063w) in S.cerevisiae are also homologous to the Nem1p C-terminal domain (Figure 1C; only two are shown). This suggests that the C-terminal domain of Nem1p defines a novel protein family which is evolutionarily conserved. The identity between the C-terminal domain of Nem1p and its highly related counterparts ranges between 34 and 37%, with 100% conserved blocks of 8-10 residues (Figure 1C). Within their N-terminal domains, the various members of this family do not show an overall sequence similarity, although individual members exhibit homology within this part of the protein (e.g. the N-terminal domain of YLR019w and YLL010c). NEM1 and SPO7 genetically interact with other NPC components NEM1 or SPO7 are not essential for cell viability; however, nem1::HIS3 and spo7::HIS3 mutants exhibit a slightly reduced growth rate at higher temperatures (37°C) (Figure 1B) and grow more slowly at lower temperatures (16°C) (data not shown). This non-essential phenotype enabled us to test genetically the functional interactions of NEM1 and SPO7 with other components of the NPC (Table I). As expected, cells without NEM1 or SPO7 are not viable in a genetic background of the nup84::HIS3 gene disruption, i.e. they are synthetically lethal. A complex pattern of genetic interactions was observed for the nem1::HIS3 and spo7::HIS3 mutants in relation to other members of the Nup84p complex. Whereas the combination spo7::HIS3 seh1::HIS3 yielded progeny which grow extremely slowly at 30°C when compared with the corresponding single mutant strains and are thermosensitive at 37°C, the nem1::HIS3 seh1::HIS3 pair gave viable cells with a normal growth rate (Table I). The opposite pattern was obtained for the nup85Δ allele when tested in combination with spo7::HIS3 and nem1::HIS3. Interestingly, spo7:: HIS3, but not nem1::HIS3, was synthetically lethal with the nup188::HIS3 disruption allele (Table I). NUP188 was often found in sl screens with nucleoporin mutants (Nehrbass et al., 1996; Zabel et al., 1996; Teixeira et al., 1997) and is linked predominantly to structural components of the NPC including Pom152p, the only known transmembrane nucleoporin in yeast (Wozniak et al., 1994; Nehrbass et al., 1996). However, no genetic interaction was detected between disruption alleles of POM152 and SPO7 (Table I). Finally, no synthetic lethal relationship was found between SPO7 and NEM1, since the combination of their disruption alleles yielded viable haploid progeny. Table 1. Synthetically lethal relationships between spo7::HIS3, nem1::HIS3 and nucleoporin mutants spo7::HIS3 nem1::HIS3 nup84::HIS3 − − nup85Δ ++ −/+ nup120::HIS3 −/+ −/+ seh1::HIS3 −/+ ++ nup188::HIS3 − ++ mtr7-1 ++ ++ pom152::HIS3 ++ n.d. nup133::HIS3 ++ n.d. nem1::HIS3 ++ ++ The genotypes of the strains are given in Table II. Double-disrupted strains are indicated by combination in the vertical and horizontal columns of single disrupted mutants. All double disruptants initially carried a URA3-containing plasmid with one of the two possible wild-type alleles. Such strains were streaked on 5-FOA-containing plates to test for synthetic lethality. The lack of growth (synthetic lethality) is expressed as ‘−’; good growth (no synthetic lethality) is expressed as ‘++’; and poor growth, only forming microcolonies, is indicated as ‘−/+’. In the latter case, the few colonies that grew on FOA plates showed a strong synergism of growth inhibition at 30 and 37°C if resteaked on a YPD plate, with respect to the corresponding single mutants. n. d., not determined. In conclusion, genetic tests revealed that NEM1 and SPO7 genetically interact with several members of the NPC implicated in NPC/nuclear envelope biogenesis. This prompted us to analyse whether Spo7p and Nem1p are also NPC proteins. Spo7p and Nem1p are nuclear envelope/ER membrane proteins To analyse the functional relationship between Spo7p, Nem1p and nucleoporins, we determined their subcellular location by tagging both proteins with the green fluorescent protein (GFP). Nem1p-GFP and Spo7p-GFP fusion constructs were expressed, under their authentic promoters, in the nem1::HIS3 and spo7:HIS3 mutants, respectively. Nem1p-GFP and Spo7p-GFP were able to complement the lethal phenotype of the corresponding sl mutants, sl235 and sl269, respectively, and were therefore functional (data not shown). Figure 2.GFP-tagged Spo7p and Nem1p exhibit a nuclear membrane/ER localization. (A) Subcellular localization of Spo7p-GFP. SPO7-GFP was expressed from a low copy (ARS/CEN) or high copy (2μ) number plasmid transformed into the spo7::HIS3-disrupted strain. (B) Subcellular localization of GFP-Nem1p. GFP-NEM1 expressed under the control of the NOP1 promoter and from an ARS/CEN plasmid was transformed into a nem1::HIS3 homozygous diploid strain. Download figure Download PowerPoint Although the expression of Spo7p-GFP was low (as judged by Western blotting using anti-GFP antibodies), a weak nuclear envelope staining was observed in the fluorescence microscope, which was significantly enhanced when Spo7p-GFP was expressed from a high copy plasmid (Figure 2A). Interestingly, this ring-like staining was not punctate but smooth, which is more typical of a nuclear membrane/ER distribution (Preuss et al., 1991). Furthermore, a discontinuous labelling close to the plasma membrane was observed which could be the underlying ER. Indeed, when a double staining experiment was performed using antibodies against Sec61p, an ER membrane protein, both signals overlapped (data not shown). Finally, Spo7p-GFP did not cluster in the nup133 deletion mutant, where bona fide nucleoporins cluster (Doye et al., 1994; Pemberton et al., 1995) (data not shown). We therefore conclude that Spo7p localizes to both the nuclear membrane and the ER, but is not associated with NPCs. However, we cannot exclude that a minor fraction of Spo7p interacts with nuclear pores (see also Discussion). Figure 3.Spo7p and Nem1p have characteristics of integral membrane proteins. (A) Schematic representation of the putative membrane-spanning sequences within Spo7p and Nem1p, whose primary sequences are schematically drawn from the N-terminal to the C-terminal end. Dark boxes represent the hydrophobic stretches. Below the dark boxes, the amino acid sequence from the hydrophobic stretches, separated by dashes from the more charged residues, is given. The hatched box within Nem1p represents its highly conserved C-terminal domain. Also shown is the Kyte-Doolittle hydrophobicity plot for Spo7p and Nem1p (Kyte and Doolittle, 1982). The window size used is 15 amino acids. (B) Spo7p and Nem1p behave biochemically as integral membrane proteins. As described in Materials and methods, a crude membrane fraction derived from cells expressing either Spo7p-ProtA or Nem1p-ProtA was extracted with buffer, 1% Triton X-100 (TX-100), salt (1 M NaCl) and pH 11.5 (0.1 M sodium carbonate). After ultracentrifugation, equivalent amounts of the insoluble pellet (P) and the supernatant (S) were analysed by SDS-PAGE followed by Western blotting using anti-ProtA, anti-Sec61p or anti-Nup85p antibodies. Download figure Download PowerPoint The expression of NEM1-GFP under its authentic promoter and from a centromeric plasmid was too low to detect a distinct GFP fluorescence signal in the cells. We therefore constructed an N-terminal GFP-NEM1 fusion under the control of the NOP1 promoter and expressed from a centromeric plasmid. This fusion protein could complement sl235 and, when localized in a nem1::HIS3-disrupted diploid, exhibited a weak nuclear envelope and peripheral ER staining (Figure 2B). This suggested that Nem1p also localizes to the nuclear/ER membrane. Hydropathy plot analysis indicated that Nem1p and Spo7p contain potential membrane-spanning sequences (Figure 3A). Interestingly, the predicted transmembrane domains of Spo7p and Nem1p are two closely spaced hydrophobic stretches, separated by a lysine and proline in the case of Nem1p and a longer, 14 residue hydrophilic sequence in the case of Spo7p (Figure 3A). In order to test whether they are integral membrane proteins, Nem1p and Spo7p were tagged with protein A (see Materials and methods). This yielded functional fusion proteins able to complement the corresponding sl mutants (data not shown). When the spheroplasts were lysed in a buffer devoid of detergent, neither Nem1p-ProtA nor Spo7p-ProtA were recovered in the soluble supernatant where most of the cellular proteins partition (data not shown). However, the same lysis buffer containing 1% Triton X-100 efficiently extracted both Nem1p-ProtA and Spo7p-ProtA. To show whether Nem1p and Spo7p are membrane associated or integral membrane proteins, a crude membrane fraction was prepared and extracted with a variety of reagents (Figure 3B). Following ultracentrifugation, the insoluble pellet (P) and soluble supernatant (S) were analysed by Western blotting using anti-ProtA antibodies. For control reasons, we also followed Sec61p, an integral ER membrane protein, and Nup85p, a component of the NPC. Detergent-free buffer or buffer containing 1 M salt did not release Nem1p-ProtA or Spo7p-ProtA. Addition of 1% Triton X-100 solubilized most of Nem1p-ProtA and Spo7p-ProtA. On the other hand, incubation of the membrane fraction in pH 11.5 (0.1 M sodium carbonate) buffer did not extract Nem1p-ProtA and Spo7p-ProtA. As expected, the integral membrane protein Sec61p, but not Nup85p, was resistant to pH 11.5 extraction. Taken together, these results indicate that Nem1p and Spo7p behave like integral membrane proteins. NEM1 and SPO7 are essential for correct nuclear morphology Cells lacking the NEM1 or SPO7 gene do not exhibit defects in nucleocytoplasmic transport pathways, as tested for nuclear uptake of a nuclear localization signal (NLS)-containing import substrate and nuclear export of mRNA (data not shown). We therefore looked for alterations in nuclear pore and nuclear membrane organization in these mutants by following the location of a nuclear pore reporter Nup49p-GFP (Belgareh and Doye, 1997; Bucci and Wente, 1997) (Figure 4A). This revealed a strikingly different pattern of Nup49p-GFP distribution. Instead of an exclusive punctate nuclear envelope staining typical for wild-type cells, Nup49p-GFP was found in many foci scattered throughout the cytoplasm of spo7 and nem1 mutants. Often these foci appeared to be aligned on a ribbon-like array, which either surrounded the vacuole or was underlying the plasma membrane. To exclude that only Nup49p-GFP mislocalized in nem1 and spo7 mutants, we purified Nup49p-ProtA from wild-type and spo7-deleted cells. In both cases, Nup49p-ProtA co-purified with the other three members of the Nup49p complex (Grandi et al., 1993), showing that its assembly is not affected (data not shown). Furthermore, when the distribution of another nucleoporin, Seh1p-GFP (Siniossoglou et al., 1996), was analysed, the same abnormal NPC staining was observed (data not shown). This all suggested that the overall NPC distribution is severely altered in nem1 and spo7 mutants. To test whether this is paralleled by an abnormal nuclear morphology, we analysed the localization of an intranuclear protein tagged with GFP, Pus1p-GFP (Hellmuth et al., 1998). A strikingly different nuclear morphology was observed in Spo7− and Nem1− cells as compared with wild-type cells (Figure 4B). Instead of being round, nuclei are irregularly shaped and elongated, and exhibit long and thin projections. The nucleus in a non-dividing cell often consists of two lobes interconnected by a long nuclear membrane extension (Figure 4B, arrow). Figure 4.Cells disrupted for the SPO7 or NEM1 genes exhibit a drastically altered nuclear morphology. (A) In vivo localization of the nuclear pore reporter protein Nup49p-GFP in wild-type, Spo7− and Nem1− cells. The corresponding strains were obtained by transformation using the NUP49-GFP plasmid construct. Note that the Nup49p-GFP staining in the case of Spo7− and Nem1− cells is no longer exclusively punctate around a spherical nucleus, but numerous foci are seen in the cytoplasm, which often arrange on a ribbon-like structure (indicated by arrows). (B) Nuclear morphology in Spo7− and Nem1− cells as revealed by intranuclear Pus1p-GFP distribution. The corresponding strains were obtained by transformation using the PUS1-GFP construct. Note that the nucleus is no longer round-shaped in Spo7− and Nem1− cells. A typical abnormality is that two nuclear lobes in a single cell are connected by a long thin extension (indicated by an arrow). (C) Nuclear envelope morphology in Nem1− cells as depicted by overexpressed Spo7p-GFP. The nem1::HIS3 mutant was transformed with a high copy number (2μ) SPO7-GFP-containing plasmid. Spo7p-GFP depicts the nuclear membrane boundary. Arrows indicate typical nuclear envelope outgrowths observed in the nem1::HIS3 mutant. Download figure Download PowerPoint Finally, Spo7p-GFP served as a marker to depict the nuclear envelope boundary in Nem1− cells (Figure 4C). This revealed that the nuclear envelope is no longer spherical, but exhibits long protrusions extending into the cytoplasm and frequently surrounding the vacuole. We also saw repeatedly that single cells contain two nuclear compartments connected by a thin membranous filament. However, no irregular DNA staining was noticed in Spo7− and Nem1− cells (Figure 5). In single cells which appeared to contain two nuclear compartments, the DNA, depicted by Hoechst 33258 staining, either in vivo (Figure 5A) or in fixed cells (Figure 5B), was round and compact, and always restricted to one nuclear compartment. Thus, nem1 and spo7 mutants are not bi- or multinucleate, which is consistent with the fact that the cell growth and, accordingly, also nuclear division is not inhibited in these mutants. In conclusion, the nuclear membrane organization is drastically altered in yeast mutants lacking SPO7 or NEM1. The nucleus is no longer round, but exhibits long extensions which contain NPCs and an intranuclear content, but not DNA. Figure 5.DNA staining is normal in Spo7− and Nem1− cells. (A) DNA staining of the spo7::HIS3 mutant expressing the Nup49p-GFP fusion. Cells grown in selective medium at 30°C were stained in vivo with Hoechst 33258 as described in Materials and methods and inspected in the fluorescence microscope. (B) DNA staining of the spo7::HIS3 mutant expressing Pus1p-GFP. Cells were grown in selective medium at 30°C, fixed, spheroplasted and stained with Hoechst 33258 as described in Materials and methods. Note that the DNA staining is restricted to a single round nuclear compartment, typical of a wild-type strain, and does not follow the nuclear content, depicted by Pus1p-GFP, that fills the nuclear membrane extensions of the spo7::HIS3 mutant. Download figure Download PowerPoint To analyse the nuclear envelope morphology of the nem1::HIS3 and spo7::HIS3 mutants at an ultrastructural level, we performed thin section electron microscopy (Figure 6). We could often see in non-dividing cells either a single nucleus with a