The Npl4 Protein Is Highly Conserved
The essential
S. cerevisiae Npl4p protein has been conserved throughout eukaryotic evolution. Figure depicts a schematic of yeast (
S. cerevisiae and
Schizosaccharomyces pombe) and higher eukaryotic (
Caenorhabditis elegans,
Drosophila melanogaster,
Rattus norvegicus, and
Homo sapiens) Npl4 proteins. In total, identity and similarity among these proteins are ~35 and 57%, respectively. The Npl4 proteins contain two notable features. First, several perfectly conserved cysteine and histidine residues in the N terminus may represent a novel Zn
2+-binding domain that could mediate protein-DNA or protein-protein interactions. Second, the higher eukaryotic Npl4 proteins contain a C-terminal predicted Zn
2+-finger Ran-GDP–binding domain similar to that found in the human nuclear pore protein Nup153 (
Nakielny et al., 1999 
;
Yaseen and Blobel, 1999 ).
Two temperature-sensitive alleles of the
S. cerevisiae NPL4 gene have been characterized in our laboratory. The first allele,
npl4-1, was generated in the nuclear protein localization (NPL) screen (
Bossie et al., 1992 ), and the second allele,
npl4-2, was isolated from a library of temperature-sensitive yeast mutants by noncomplementation with
npl4-1 (
DeHoratius and Silver, 1996 ). Inspection of the sequence from the
NPL4 promoter, open reading frame, and 3′-untranslated region from the wild-type and mutant strains revealed that
npl4-1 harbors a G→A mutation that substitutes a serine for glycine at position 323. This glycine residue is absolutely conserved in all Npl4 homologues (Figure ), and lies within a block of amino acids that show strong conservation. The
npl4-2 allele contains a G→A mutation that places a premature stop codon 12 amino acids from the C terminus of wild-type Npl4p (Figure ).
Npl4p Localizes to Perinuclear Membranes
Previous studies have suggested that Npl4p may be a component of the nuclear pore given its localization in fixed cells by indirect immunofluorescence to the nuclear rim (
DeHoratius and Silver, 1996 ). To localize Npl4p in living yeast cells, we integrated DNA encoding a C-terminal GFP tag at the
NPL4 genomic locus. Western blotting indicated that the resulting fusion protein was full-length and replaced endogenous untagged Npl4p (Figure A, lanes 2 and 5). Viability of the resulting strain confirms the functionality of Npl4p-GFP as it is the only form of Npl4p present in the cells. By fluorescence microscopy of living yeast cells, Npl4p-GFP appears to localize mainly to the nucleus (Figure B, arrowhead) and cytoplasm, possibly with a concentration at perinuclear membranes. In some cases, Npl4p-GFP signal can also be detected in what appears to correspond to cortical membranes (Figure B, arrow). To confirm that the membrane localization of Npl4p-GFP is perinuclear, we formaldehyde fixed and DAPI stained Npl4p-GFP-expressing yeast cells. As can be seen in Figure C, the majority of Npl4p-GFP signal can be seen in a region overlapping with or around the nuclear DNA. The diffuse cytoplasmic pool of Npl4p-GFP can also be seen in these cells. Finally, to analyze the Npl4p-GFP localization pattern in more detail, we viewed live cells on a DeltaVision platform. Images were captured in 0.1-μm sections through each cell and were subjected to deconvolution to minimize background and out-of-focus signal (
Agard et al., 1989 ). After deconvolution, the presence of Npl4p-GFP at perinuclear (Figure D, arrowhead) and peripheral membranes (Figure D, arrows) was especially apparent. This perinuclear membrane signal of Npl4p-GFP is very similar to that expected for a protein that associates with ER and nuclear membranes (which are contiguous in yeast).
To biochemically confirm this Npl4p localization pattern, yeast extracts were separated into soluble and microsomal (ER/nuclear membrane) fractions (see MATERIALS AND METHODS). Immunoblotting against equal amounts of protein from each fraction with anti-Npl4 antibodies revealed that endogenous Npl4p is associated with microsomal membranes (Figure A, lane 2) and is present in soluble pools (Figure A, lane 3). This Npl4p fraction remained soluble even after a high-speed (100,000 × g) spin (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results). We estimate that ~20% of cellular Npl4p is membrane associated based on the fact that similar levels of Npl4p are found in crude (Figure A, lane 1) and microsomal (Figure A, lane 2) fractions, whereas total microsomal protein isolated was 1/5 of the total cellular protein. Npl4p-GFP displayed the same fractionation profile (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results).
Given that the Npl4p amino acid sequence does not contain any predicted transmembrane domains, we wanted to test the nature of the Npl4p interaction with microsomal membranes. Microsomal membranes derived from a wild-type yeast strain were washed with various buffers and separated into soluble (S) and pellet (P) fractions (Figure B). Immunoblotting with anti-Npl4 revealed that Npl4p could not be extracted into the soluble fraction by high salt (1 M and 2 M potassium acetate, lanes 4 and 6, respectively) and was only partially dissociated from membranes by 3 M urea (Figure B, lane 8) or 0.1 M sodium carbonate, pH 11 (Figure B, lane 10). Only in the presence of 1% Triton X-100 was Npl4p completely extracted (Figure B, lane 12). The Npl4p-interacting protein Cdc48p (Figure C) displayed a similar extraction profile as previously published (
Latterich et al., 1995 ). As controls, these same fractions were also immunoblotted with antibodies against the peripheral ER membrane protein Sec17p and integral ER membrane protein Sec62p. As expected, Sec17p dissociated from the membranes upon the addition of 3 M urea (Figure B, lane 8) and 0.1 M sodium carbonate, pH 11 (Figure B, lane 10). In contrast, Sec63p was not dissociated from microsomal membranes under any conditions except when 1% Triton X-100 was added (Figure B, lane 12). These results suggest that a subset of cellular Npl4p protein behaves as a tightly associated peripheral ER/nuclear membrane protein.
Npl4p Complexes with Ufd1p and the AAA-ATPase Cdc48p
In an effort to identify other proteins with which Npl4p might interact, we integrated DNA encoding protein A (pA) at the
NPL4 genomic locus, generating a
NPL4-pA open reading frame. Expression of a full-length fusion protein that replaced endogenous Npl4p was confirmed by Western blotting (Figure A, lanes 3 and 6). The functionality of this construct was confirmed by viability of the resulting strain. Extracts were prepared from yeast cells expressing Npl4p-pA or untagged Npl4p as a control. After incubation with IgG-Sepharose and extensive washing, bound proteins were eluted, separated by SDS-PAGE, and visualized by Coomassie blue staining (Figure C). In the Npl4p-pA sample, three major eluted proteins were observed: p100, p90, and p43 (Figure C, lane 2). In the untagged sample, only background Coomassie blue staining was observed (Figure C, lane 1). The bands were excised from the gel and subjected to mass spectral analysis for identification. p90 was identified as the Npl4p-pA fusion protein, p100 was identified as Cdc48p, and p43 was identified as Ufd1p. Ufd1p is an essential protein of unknown function that was identified in a screen for yeast mutants that stabilize an artificial ubiquitin fusion protein (the ubiquitin fusion degradation [UFD] screen) (
Johnson et al., 1995 ). The Cdc48p protein, along with its mammalian homologue p97, is a putative chaperone of the AAA-ATPase family that has been implicated in homotypic membrane fusion, ubiquitin-mediated protein turnover (including the UFD pathway), and cell cycle progression (
Moir et al., 1982 ;
Frohlich et al., 1991 ;
Acharya et al., 1995 ;
Latterich et al., 1995 ;
Rabouille et al., 1995 ;
Ghislain et al., 1996 ;
Dai et al., 1998 ). The mammalian Npl4, Ufd1, and p97/Cdc48 proteins have also been recently demonstrated to interact (
Meyer et al., 2000 ), indicating that this protein complex has been evolutionarily conserved.
Based on the interaction of Npl4p with Cdc48p, as well as the membrane localization of Npl4p, we tested whether
npl4 mutants display defects in homotypic membrane fusion. Microsomal membranes were isolated from wild-type,
npl4-1, and
npl4-2 yeast strains and then tested for their ability to fuse in a previously described in vitro homotypic membrane fusion assay (
Latterich and Schekman, 1994 ). Membranes derived from
npl4 strains were able to fuse to levels comparable to wild-type membranes (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results), suggesting that Npl4p is not required for homotypic membrane fusion.
npl4 Mutants Are Suppressed by Overproduction of Components of the Fatty Acid Desaturation Pathway and the Proteasome-associated Gene RPN4
In an effort to uncover the primary defect of
npl4 strains, a screen was performed to identify yeast genes that, when overexpressed, can rescue
npl4-1 temperature sensitivity. The
npl4-1 strain was transformed with a high-copy (2 μ
URA3) yeast genomic library (gift of C. Connelly and P. Hieter) and screened for growth at the nonpermissive temperature (30°C). One gene isolated from this screen,
OLE1, encodes the yeast Δ9-fatty acid desaturase, which is required for all de novo synthesis of UFAs (
Stukey et al., 1990 ). As shown in Figure A (top), the temperature sensitivity of
npl4-1 at 30°C is strongly rescued by 2 μ
OLE1 as compared with the empty vector control. However,
npl4-1 growth at the higher temperature (36°C) is not rescued, indicating that
OLE1 is only able to complement
npl4-1 temperature sensitivity at intermediate temperatures. Temperature sensitivity of
npl4-2 at 36 and 37°C is also rescued by 2 μ
OLE1 (Figure A, bottom), indicating that this effect is not allele specific.
The other gene isolated from this
npl4-1 high-copy suppressor screen,
MGA2, encodes a transcription factor that has been shown to activate the transcription of
OLE1 (
Zhang et al., 1999 ). Mga2p and the redundant transcriptional activator Spt23p have recently been shown to be made as membrane-bound precursor proteins, which are then cleaved in a proteasome-dependent manner from the membrane (
Hoppe et al., 2000 ). It is hypothesized that cleavage from the membrane is required for subsequent activation of
OLE1 transcription by Mga2p and Spt23p. Interestingly, the two
MGA2 clones isolated in our screen contained identically truncated forms of
MGA2 lacking the coding sequence for the C-terminal 199 amino acids. The site of truncation, which eliminates the transmembrane domain (indicated by an arrow in the Mga2p schematic in Figure C) likely leads to expression of a soluble, truncated form of Mga2p. As with
OLE1, the truncated
MGA2 clone can rescue
npl4-1 and
npl4-2 temperature sensitivity at intermediate temperatures (Figure A, top and bottom). When tested directly, full-length
MGA2 was able to suppress the temperature sensitivity of both
npl4-1 and
npl4-2 similarly to the truncated form of
MGA2, although this suppression was slightly weaker for
npl4-1 (Figure A top). In addition, the redundant gene
SPT23 was tested for high-copy suppression of
npl4 temperature sensitivity (
Zhang et al., 1997 ;
Zhang et al., 1999 ). In the case of
npl4-1,
SPT23 could only weakly suppress the growth defect at the intermediate temperature of 30°C (Figure A, top). In contrast,
npl4-2 mutants were almost completely rescued by
SPT23 (Figure A, bottom).
As additional evidence for the genetic interaction of NPL4 and SPT23, we have isolated a transposon-insertion allele of SPT23 that acts as a dominant extragenic suppressor of npl4-1 temperature sensitivity at 30°C but not higher temperatures (see Figure B and MATERIALS AND METHODS). Sequencing of transposon and flanking genomic DNA revealed that the transposon insertion results in the placement of an in-frame valine codon and stop codon 2130 nucleotides (corresponding to 710 amino acids) into the coding sequence of SPT23. The nature of this insertion would prevent expression of the predicted ankyrin repeats and transmembrane domain (see Spt23p schematic in Figure C). This finding indicates that single-copy expression of a truncated, soluble form of Spt23p can extragenically suppress npl4-1 temperature sensitivity at 30°C.
The results of these genetic screens suggest that the temperature sensitivity of npl4 mutants can be mitigated by increasing the levels of the OLE1 gene—either directly, by overexpressing OLE1 itself, or indirectly, by overexpressing or truncating (and presumably activating) transcription factors that direct OLE1 expression. Given that the primary function of Ole1p is to produce UFAs, we sought to determine whether npl4 mutants could be rescued by supplementing their growth media with UFAs. Indeed, growth of npl4-1 and npl4-2 strains at intermediate temperatures (30 and 36°C, respectively) is restored by supplemented palmitoleic (16:1) and oleic acid (18:1; Figure D). This effect was not due to the detergent used to solubilize the UFAs (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results). The ole1Δ strain was used as a control at all temperatures because its growth relies exclusively on the presence of supplemented UFAs.
Through the course of our studies, we have also discovered a genetic interaction between
NPL4 and the proteasome regulator
RPN4/
SON1/
UFD5. RPN4 was originally identified in our laboratory as an extragenic suppressor of C-terminal mutants in
SEC63/
NPL1 (
Nelson et al., 1993 ) and was also isolated in the same genetic screen as
UFD1 (
Johnson et al., 1995 ). In addition,
RPN4 has been shown to physically associate with the proteasome (
Fujimuro et al., 1998 ) and to regulate proteasome gene transcription (
Mannhaupt et al., 1999 ). When expressed in high copy,
RPN4 strongly rescues the temperature sensitivity of both
npl4-1 and
npl4-2 mutant strains as compared with empty vector DNA (Figure A). In the case of
npl4-1,
RPN4 rescues growth at the intermediate temperature of 30°C (Figure A, top), whereas in the case of
npl4-2,
RPN4 is capable of rescuing growth at both 36 and 37°C (Figure A, bottom). Furthermore,
npl4-1rpn4Δ double mutants display a synthetic slow-growth phenotype at 25°C compared with wild type and both single mutants alone (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results).
Npl4p Is Required for OLE1 Expression
The genetic data presented above led us to the hypothesis that Npl4p may be required for the proteasome-dependent processing/activation of the Mga2p and Spt23p transcription factors and subsequent
OLE1 expression (
Hoppe et al., 2000 ). The isolation of truncated forms of
MGA2 and
SPT23 as suppressors of
npl4 temperature sensitivity provided especially strong support for this hypothesis. As a first test, we asked whether
OLE1 transcript levels are reduced in
npl4 mutant cells compared with those of a control transcript (
ACT1). Northern blot analysis was performed against total RNA isolated from wild-type,
npl4-1, and
npl4-2 strains before and after a shift to the nonpermissive temperature (37°C). As shown in Figure ,
OLE1 transcript levels are normal in both
npl4-1 and
npl4-2 cells at permissive temperature (Figure , compare lanes 3, 6, and 9). However,
OLE1 levels are dramatically decreased in these mutants after a 60-min shift to 37°C (Figure , lanes 7 and 10). In contrast,
OLE1 expression is stimulated in wild-type cells after a shift to 37°C (Figure , lane 4). The
npl4-1 and
npl4-2 strains were capable of repressing
OLE1 expression to a similar extent as wild-type cells (Figure , lanes 2, 5, and 8). To quantitate the change in
OLE1 expression, relative
OLE1/
ACT1 signal was calculated (see MATERIALS AND METHODS; Figure , bottom).
Given the striking effect of NPL4 loss of function on OLE1 expression, we determined whether the NPL4 associated genes, CDC48, UFD1, and RPN4, are also required for OLE1 expression. As a test of the requirement for the Npl4p-interacting protein Cdc48p, Northern blot analysis was performed against total RNA isolated from the temperature-sensitive cdc48–3 strain. As with npl4-1 and npl4-2, OLE1 levels were normal at the permissive temperature of 25°C in cdc48-3 cells (Figure , compare lanes 3 and 12). However, OLE1 mRNA became undetectable after a 60-min shift to the nonpermissive temperature of 37°C (Figure , lane 13). To determine whether the Npl4p-interacting protein Ufd1p is also required for OLE1 expression, we monitored OLE1 mRNA levels in the ufd1-1 mutant strain. No significant defect in OLE1 expression was apparent in ufd1-1 cells at 25°C as compared with wild-type cells (Figure , compare lanes 3 and 15). Interestingly, the ufd1-1 strain had significantly lower levels of OLE1 mRNA after a 60-min shift to 37°C (Figure , compare lanes 4 and 16), despite the fact that this strain is not temperature sensitive. Finally, we tested the requirement for the RPN4 gene in OLE1 expression. OLE1 transcript levels were normal in the rpn4Δ strain at 25°C (Figure , compare lanes 3 and 18). Like the ufd1-1 strain, rpn4Δ cells are not temperature sensitive; however, after a shift to 37°C for 60 min, OLE1 levels were significantly decreased in the rpn4Δ strain as compared with wild type (Figure , compare lanes 4 and 19). The cdc48-3, ufd1-1, and rpn4Δ mutants were able to repress OLE1 mRNA expression in the presence of UFAs, although not quite as strongly as wild-type cells (Figure , lanes 2, 11, 14, and 17).
Npl4p Is Required for Efficient Processing of Mga2p and Spt23p
To more directly test our hypothesis that Npl4p function is upstream of Mga2p and Spt23p processing and activation, we expressed galactose-inducible N-terminally GFP-tagged Mga2p and Spt23p proteins in wild-type and npl4 mutant cells. At various time points into fusion protein induction, cells were collected and whole cell extracts were subjected to anti-GFP Western blot analysis. Within 60 min of induction in wild-type cells, both full-length and processed forms of GFP-Mga2p and GFP-Spt23p are detectable at significant levels (Figure , A and B, respectively, lanes 1–4). In contrast, very little processed GFP-Mga2p was detectable in npl4-1 and npl4-2 cells even after a 120-min induction (Figure A, lanes 7 and 10, respectively). Similarly, processed GFP-Spt23p was not detectable in npl4-1 and npl4-2 cells (Figure B, compare lanes 7 and 10 to lane 4). It should be noted that these experiments were performed at 25°C, indicating that efficient GFP-Mga2p and GFP-Spt23p processing is compromised in these strains even at the permissive temperature.
We also tested whether the Npl4-associated genes CDC48, UFD1, and RPN4 are required for efficient GFP-Mga2p and GFP-Spt23p processing. A strain harboring the temperature-sensitive cdc48-2 mutation failed to accumulate processed GFP-Mga2p and GFP-Spt23p, similar to npl4 cells (Figure , A and B, lane 13). In addition, ufd1-1 and rpn4Δ cells were unable to accumulate processed forms of these fusion proteins (Figure , A and B, lanes 16 and 19, respectively). We noted from this Western analysis that the unprocessed form of GFP-Mga2p and GFP-Spt23p in these mutant strains was often accompanied by accumulation of higher molecular weight species (see asterisks in Figure , A and B).
We then observed these cells by fluorescence microscopy to determine the subcellular localization of GFP-Mga2p and GFP-Spt23p in npl4 and associated mutants. The results obtained with GFP-Spt23p–expressing cells are shown in Figure C. Strikingly, although GFP-Spt23p was mostly nucleoplasmic after 120 min of induction in wild-type cells, it was tightly associated with perinuclear envelopes in npl4-1, npl4-2, cdc48-2, ufd1-1, and rpn4Δ strains. GFP-Mga2p showed a similar localization pattern in these strains (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results). These results suggest that lack of processing of these fusion proteins in these mutant strains corresponds to an inability of this protein to dissociate from ER/nuclear envelope membranes.
Finally, based on our observation of higher molecular weight species of GFP-Mga2p and GFP-Spt23p in npl4 and associated mutant strains (Figure , A and B, asterisks), we tested whether these fusion proteins are ubiquitinated in the npl4-1 mutant. To this end, galactose-induced GFP-Mga2p and GFP-Spt23p proteins were immunoprecipitated from whole cell extracts derived from wild-type or npl4-1 yeast expressing a Myc epitope-tagged ubiquitin. Western analysis of bound fractions revealed significant levels of myc-immunoreactive proteins with sizes larger than the unprocessed GFP-Mga2p and GFP-Spt23p fusion proteins in both wild-type and npl4-1 cells (Figure D, lanes 6, 8, 14, and 16). Similar results were obtained with the npl4-2 mutant strain (Hitchcock, Krebber, Frietze, Lin, Latterich, and Silver, unpublished results). These data suggest that ubiquitination of GFP-Mga2p and GFP-Spt23p is not blocked in npl4 mutant cells.
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