Identifying Genes Required for Cell or Nuclear Fusion
Many of the genes currently known to be involved in mating are induced by pheromone; moreover, the mechanisms of plasma and nuclear membrane fusion are likely to involve membrane-bound proteins. To identify pheromone-inducible membrane proteins, we extended an approach used by Heiman and Walter (2000)
to screen published databases to identify putative pheromone-induced genes with predicted transmembrane domains. To increase the range of potential genes, we identified the set of genes showing at least threefold induction upon exposure to pheromone at various times after treatment (Roberts et al., 2000
). The presence of transmembrane domains was predicted using the SOSUI program (Hirokawa et al., 1998
) and TMHMM program (Krogh et al., 2001
). In total, 55 pheromone-induced genes were predicted to encode transmembrane proteins. Half of the candidates had been characterized previously and were demonstrated to have roles in cell wall synthesis and/or mating, including PRM1
, and FUS1
. The effects of deletions in the 27 remaining genes not described previously as having a role in mating were assayed for potential effects on mating by using a quantitative microscopic mating assay (Gammie et al., 1998
). This set included six genes identified previously as encoding potential pheromone-induced membrane proteins (Heiman and Walter, 2000
). Eight mutants showed a significant cell or nuclear fusion defect (Supplemental ). However, only one deletion mutant exhibited a strong defect (YPL192C/PRM3
) and was characterized further.
Prm3p Is Required for Nuclear Membrane Fusion
To understand the nature of the mutant phenotype, prm3 zygotes were examined microscopically for mating defects. Karyogamy mutants are classified as either unilateral or bilateral; unilateral mutants are defective when mated to wild-type cells, bilateral mutants show a defect only when both mating partners are mutant. The prm3 mutant zygotes showed a strong bilateral karyogamy defect (A and ). No defect was apparent in unilateral matings, indicating that Prm3p need be expressed in only one cell for nuclear fusion to occur (). The nuclear fusion defect in prm3 mutant zygotes was complemented by a plasmid carrying a copy of PRM3, as well as N-terminal FLAG and GFP-tagged PRM3 (). Epitope tags at the C terminus of Prm3p resulted in nonfunctional constructs, indicating that the C terminus is important for protein function.
Figure 1. Prm3p is required for nuclear membrane fusion. (A) prm3Δ mutant zygotes have closely apposed nuclei (stained with DAPI) that do not fuse (MS7590 × MS7591). (B) prm3Δ mutant zygote with 3X-GFP-HDEL expressed around both nuclei. (more ...)
prm3 is a unilateral karyogamy mutant
The close apposition of the nuclei in prm3
zygotes is consistent with a class II nuclear fusion defect (Kurihara et al., 1994
), in which the nuclei have congressed but nuclear envelope fusion is blocked. To detect a defect in membrane fusion, we examined prm3
zygotes in which the nuclear envelope lumen was labeled with 3XGFP-HDEL and the spindle pole bodies were labeled with Spc42p-red fluorescent protein (RFP) (Melloy et al., 2007
). Consistent with a defect in nuclear envelope fusion, the two spindle bodies moved close together, but distinct nuclear envelopes remained surrounding the two unfused haploid nuclei (B). In wild-type zygotes, the nuclear envelopes fuse quickly to form a diploid nucleus (C). We conclude that Prm3p is required for nuclear envelope fusion.
Prm3p Is a Pheromone-induced Peripheral Membrane Protein
To understand the role Prm3p plays in nuclear membrane fusion, we investigated its structure, expression, and localization within the cell. Prm3p is a small protein of 133 residues, with a predicted transmembrane domain at the carboxy terminus between residues 107-127. Comparison with homologous genes from related yeast species (Cliften et al., 2003
; Kellis et al., 2003
) showed that the carboxy-terminal region of Prm3p is highly conserved (). This region includes the sole predicted transmembrane domain. The presence of conserved charged residues spaced three or four residues apart in the amino-terminal region suggested a coiled-coil structure that may interact with other proteins. In the middle of the protein are two small highly conserved regions consisting of positively charged residues ().
Figure 2. The hydrophobic C-terminal region of Prm3p is highly conserved. (A) Comparison of the Prm3p protein sequence between S. cerevisiae, Saccharomyces bayanus, Saccharomyces mikatae, and Saccharomyces paradoxus. Figure modified from the Fungal Sequence Alignment (more ...)
The upstream region of PRM3 contains a putative pheromone response element, consistent with its regulation by pheromone. To confirm the results from the genome-wide study of pheromone-induced genes, expression of FLAG-Prm3p in both mitotic and pheromone-treated cells was determined using Western blot analysis. Expression of FLAG-Prm3p was only detected in pheromone-treated cells (A), consistent with a role in karyogamy.
Figure 3. Prm3p is a pheromone-induced peripheral membrane protein. (A) Western blot showing the induction of FLAG-Prm3p upon addition of α-factor. Protein was isolated from cells with (MS7638) or without (MS7590) FLAG-PRM3. Blots were probed with anti-FLAG (more ...)
Although membrane fusion is typically catalyzed by cytoplasmic proteins, all of the proteins identified previously to be strongly required for nuclear membrane fusion (Kar5p, Kar2p, and Kar8p) are located within the nuclear envelope/ER lumen. Initially predicted to have a single transmembrane domain, Prm3p could be oriented with its amino-terminal domain either exposed to the cytoplasm or contained within the lumen of the membrane. To determine the orientation of Prm3p, membrane fractions isolated from cells expressing FLAG-Prm3p were exposed to exogenous protease with and without detergent. Exposed portions of the protein are sensitive to proteases, whereas proteins contained within the lumen are resistant. The majority of FLAG-Prm3p was sensitive to protease in the absence of detergent (B). Kar2p, a lumenal protein, remained resistant to protease, demonstrating that the membranes remained intact throughout the course of the experiment (B). Comparison of FLAG-Prm3p and Kar2p levels showed that FLAG-Prm3p was degraded with a half-life of ~3 min. Approximately 15% of the protein was not digested within the course of the experiment, due either to protection within a protein complex or the formation of a small amount of inside-out vesicles that protected a fraction of the protein. After addition of detergent to disrupt the membranes, lumenal proteins became fully accessible to proteases, and both proteins were rapidly degraded. We conclude that the Prm3p is oriented such that it is exposed on the outside of the nuclear envelope and not contained within the lumen.
Protein extraction was performed to determine whether Prm3p is an integral membrane protein. Crude membrane fractions were treated with various reagents to extract Prm3p from the membrane. As controls, we also examined the extraction of Sec34p, a peripheral membrane protein and Sec22p, an integral membrane protein (C). Typically, peripheral proteins can be extracted with salt or high pH, which disrupt protein–protein interactions, whereas integral membrane proteins require detergents for extraction. As observed previously, the peripheral protein Sec34p was partially extracted under all conditions, but most strongly by high pH (VanRheenen et al., 1999
). The integral membrane protein Sec22p was not extracted by either 1 M NaCl or high pH. Sec22p was partially extracted by detergent alone and more completely by detergent plus 1 M NaCl. Unlike Sec22p, Prm3p was substantially extracted by 1 M NaCl and not at all by detergent alone. The partial extraction of Prm3p by 1 M NaCl was similar to Sec34p, and all three proteins were more completely extracted by detergent plus 1 M NaCl (C). Unlike Sec34p, Prm3p was not extracted by high pH. However, Prm3p has an isoelectric point of 11.1, and many proteins are not soluble near their isoelectric point. Furthermore, Sec72p, another peripheral ER membrane protein is also not extracted at high pH (Feldheim and Schekman, 1994
; Kaiser et al., 2002
). Together with the protease protection data, the protein extraction data indicate that Prm3p is actually a peripheral membrane protein. Unlike the other proteins required for nuclear membrane fusion, Prm3p seems to reside entirely outside of the ER lumen.
Prm3p Is Localized to the Cytoplasmic Face of the Nuclear Envelope
Prm3p may reside on the cytoplasmic or nuclear face of the nuclear envelope. Fusion of GFP or the FLAG epitope to the N terminus resulted in a fully functional protein (). Both the GFP fusion (– and ) and the FLAG-tagged protein ( and ) localized to the nuclear envelope when expressed in either mating or mitotic cells. Similar localization results for the full-length protein in mitotic cells were reported previously (Beilharz et al., 2003
). Previously, Prm3p was reported to be a “tail-anchored” integral membrane protein resident on the inner nuclear membrane (Beilharz et al., 2003
). In that study, GFP was fused to a truncated form of Prm3p lacking the hydrophobic C-terminal domain and was found to localize in the nucleus. Mutation of a putative nuclear localization sequence (NLS) in the truncated protein resulted in mislocalization throughout the cytoplasm. In addition, mutations that disrupt nuclear import (rna1-1
) by failing to regulate the Ran-GTPase caused increased localization of full-length GFP-Prm3p to more peripheral ER (Beilharz et al., 2003
; King et al., 2006
). Based on these results, the authors concluded that native full-length Prm3p is an inner nuclear membrane protein. However, localization to the inner nuclear envelope would be surprising, given its role in nuclear envelope fusion. As we show in the next section, the carboxy-terminal region is critical for Prm3p function and its deletion leads to a nonfunctional protein. When the same NLS mutation was introduced into full-length GFP-Prm3p, or the region containing the putative NLS was deleted, the proteins localized to the nuclear envelope (F) and supported wild-type levels of nuclear fusion during mating (G). Thus, it is unlikely that the putative NLS plays a critical role in Prm3p function. In addition, mutations that affect nuclear import are likely to have a major effect on the localization of many proteins and the mislocalization of Prm3p may result from indirect effects on nuclear structure.
Figure 4. Prm3p is on the cytoplasmic face of the nuclear envelope. (A) Immuno-EM of the nucleus in a shmoo expressing GFP-Prm3p. Cells (MS7590 with MR5062) were induced with α-factor, fixed with high-pressure freezing, embedded in plastic, and sectioned. (more ...)
Figure 6. Point mutations in the C terminus affect protein function and localization. (A) Diagram depicting point mutations and deletions in Prm3p. Deletions of the N terminus, the C terminus, and two charged conserved regions in the interior of the protein are (more ...)
Figure 8. Prm3p localization at the SPB is partially dependent on Kar5p. Coimmunofluorescence of GFP-Prm3, labeled with anti-GFP, and the spindle pole body, labeled with anti-tub4 against γ-tubulin. In A–C, top panels shows a merge of GFP-Prm3p (more ...)
Figure 7. Prm3p is enriched at the SPB. (A) Prm3p shows perinuclear staining with one or more concentrated dots. The first panel shows FLAG-Prm3p localization, the second panel is Spc42-GFP (SPB), the third panel shows the nucleus (stained with DAPI), the fourth (more ...)
Given its role in nuclear fusion, it is likely that Prm3p is located on the cytoplasmic face of the nuclear envelope. We therefore used two approaches to determine on which face of the nuclear envelope Prm3p resides. Because light microscopy does not provide sufficient resolution to distinguish between the outer and inner nuclear membranes, we used immunoelectron microscopy to localize GFP-Prm3p in shmoos. GFP-Prm3p was detected with mouse anti-GFP and F(ab′)2 anti-mouse conjugated to 10-nm gold particles. Gold particles were enriched in the vicinity of the nuclear envelope, as well as sporadically elsewhere in the cell (A). The positions of the gold particles relative to the center of the NE lumen were measured (B); positive numbers indicate distances toward the cytoplasm, negative numbers indicate distances toward the nucleus. The maximum distance between a gold particle and the center of the ER lumen should be roughly 50 nm, including the length of the primary and secondary antibodies, Prm3p itself, and distance from the membranes to the center of the lumen. Gold particles >80 nm away from the center of the lumen were not measured and were taken to be background staining. In total, 79 gold particles within 80 nm on either side of the nuclear envelope were observed in 36 different cells. In addition, 104 gold particles whose distance from the NE lumen was >80 nm were observed scattered over the entire cross sections of the cells. We estimate that the region of the nuclear envelope showed ~20-fold enrichment in gold particles. The histogram (B) shows that the gold particles were highly enriched in a region centered on the outer nuclear membrane. The number of gold particles located within 50 nm of the center of the NE nuclear envelope was significantly higher on the outer membrane than on the inner nuclear membrane (p = 0.0022; chi-square test). Assuming that the particles are symmetrically distributed about the outer membrane, then the excess of gold particles on the inner membrane (11 particles) would constitute no more than ~15% of the total.
For an independent test of Prm3p localization, we used live cell microscopy to measure the relative kinetics of transfer of Prm3p between nuclei during nuclear fusion. Because outer nuclear membrane fusion occurs before inner nuclear envelope fusion (Melloy et al., 2007
), if Prm3p is located on the cytoplasmic face of the nucleus, then it should transfer to the recipient nucleus before a nucleoplasmic protein is able to transfer. Conversely, if Prm3p is only located on the inner nuclear membrane, then transfer of Prm3p could only occur after inner nuclear membrane fusion, at the same time as the nucleoplasmic protein. For this experiment, time-lapse microscopy was performed on wild-type zygotes in which one parent expressed GFP-Prm3p and the other parent expressed Pap1p-mCherryFP as the nucleoplasmic marker. Images of zygotes were taken at 1-min (n = 4) or 30-s intervals (n = 15) for 15 min, starting immediately after completion of cell fusion. In the example shown, GFP-Prm3p began to transfer to the recipient nucleus after 4 min, whereas the nucleoplasmic marker did not transfer until after 5 min (C). Fluorescence intensity in the recipient nuclei was measured and graphed as a percentage of total fluorescence in the zygotes (, D and E). The median delay between GFP-Prm3p and Pap1p-cherryFP transfer at half-maximal transfer was ~45 s (n = 19). The median delay for the initial time of entry was ~23 s. In four of 19 zygotes, no delay was observed; transfer of Pap1p-mCherryFP before GFP-Prm3p was not observed. Thus, both the immuno-EM and live cell microscopy demonstrate that Prm3p is primarily located on the cytoplasmic face of the nuclear envelope.
In contrast to the short-term experiments, longer term time-lapse microscopy provided a likely explanation for why prm3 mutants are bilateral, that is, why Prm3p is not required to be expressed in both mating cells for nuclear fusion to continue. GFP-Prm3p was expressed from the GAL1 promoter, and cells were mated to a kar1-1 strain to prevent nuclear congression. Matings were performed on glucose media to shut off synthesis of GFP-Prm3p. As expected, GFP-Prm3p was restricted to one nucleus immediately after cell fusion. After 30 min, GFP-Prm3p was detected on the other nucleus, eventually reaching comparable levels (A). By 60 min, the zygote has reentered the mitotic cycle, and two nuclei have moved together near the incipient bud. Thus, one explanation for the prm3 bilateral phenotype is that Prm3p is only needed in one partner because the protein can shuttle between the two nuclei. Interestingly, when GFP-Prm3p was expressed from the GAL1 promoter in mitotic cells before mating, it caused striking alterations in the shape of the nuclear envelope (B), possibly indicating a role for Prm3p in the structure of the nuclear membranes during karyogamy.
Figure 5. Prm3p Shuttles Between Nuclei. (A) Time-lapse microscopy of GFP-Prm3p in a kar1-1 zygote. Microscopy began 90 min after mixing a and α cells; images were taken every 5 min. Fluorescence can be seen the second nucleus at t = 35 min. (B) Two samples (more ...)
The C-Terminal Region of Prm3p Is Required for Function, Stability, and Localization
To identify the regions of Prm3p required for nuclear envelope-specific localization and function, we analyzed the effects of point and deletion mutations in the gene. To start, we conducted an in vitro mutagenesis screen of PRM3 to identify point mutations that cause defects in mating. GFP-PRM3 and FLAG-PRM3 on centromere plasmids were mutagenized using error-prone PCR, transformed into a prm3Δ strain, and the transformants were mated to a prm3Δ strain at 23, 30, and 37°C to identify mutants unable to form diploids. Eight point mutations (indicated in and 6A and ) were identified, all located within the conserved hydrophobic C-terminal region. Two point mutations caused temperature-sensitive defects, losing nuclear fusion function at 37°C; growth and cell fusion were not affected. Thirteen nonsense mutations were identified by sequencing and not studied further.
All eight point mutations affected the function, stability, and localization of Prm3p to different extents. Nuclear fusion efficiency decreased to 10–25% of wild type (G). Protein levels in prm3-1, prm3-2, prm3-3, prm3-5, and prm3-8 strains were greatly decreased (C). Prm3p was mislocalized in prm3-2, prm3-3, prm3-5, prm3-6, and prm3-8 cells (B). Mislocalization of the mutant protein and decreased protein levels were correlated, except for mutant prm3-6, in which the protein ran at a lower molecular weight. Only in mutant prm3-4 was the protein stable and localized normally.
Two point mutants, prm3-1 and prm3-7, were temperature sensitive. In both strains, protein function was only slightly reduced at 23°C; nuclear fusion efficiency decreased with increased temperature (G). Protein levels in both mutants decreased and Prm3p became increasingly mislocalized with increased temperature (, D and E). However, Prm3p became mislocalized in the prm3-7 mutant at a lower temperature; some mislocalization was seen at 23°C. The presence of stable but mislocalized protein at 23°C suggests that mislocalization of the Prm3p protein by itself does not lead to destabilization. The isolation of point mutations causing defects in nuclear fusion only in the conserved C-terminal region underscores the essential role of this part of the protein for stability, localization, and function.
We used a directed mutagenesis approach to probe the function of the other conserved regions of Prm3p. For example, each of the conserved charged residues in the N-terminal region suggestive of a coiled-coil structure were changed to alanine ( and , prm10-prm17); none affected Prm3 function, localization, or stability (data not shown). Because it was possible that multiple residues would need to be mutagenized to cause a defect in function or localization, a deletion of the entire N terminus (residues 1-46) was constructed. Deletion of the N-terminal region (NΔ) had no affect on protein function, stability, or localization (, F and G). The internal section of Prm3p contains two conserved regions largely made up of positively charged residues. Deletion of region I3 (residues 86-96) had a significant effect on nuclear fusion efficiency and protein stability (, F and G). Deletion of region I2 (residues 63-76), which includes the putative NLS sequence, had no effect on function or localization (, F and G). As expected, deletion of the C-terminal region (residues 104-128) had severe affects on protein function and localization. Together, the mutational analysis indicates that the C-terminal half of Prm3p is the most critical part of the protein, responsible for function, localization, and stability.
Prm3p Is Concentrated at the SPB and Interacts with Kar5p
Although Prm3p localized to the nuclear envelope in shmoos, immunofluorescent detection of both FLAG-Prm3p and GFP-Prm3p showed distinctly nonuniform staining. In 78% of shmoos, one or more concentrated dots of Prm3p staining were present along the nuclear envelope (A). One of the Prm3p dots was adjacent to or colocalized with the SPB in 61% of wild-type shmoos (A) and 96% of zygotes (n = 151; Kar5p, another protein important in karyogamy, is localized adjacent to the SPB (Beh et al., 1997
). When Kar5p was overexpressed, Prm3p localization at or adjacent to the SPB increased to 93% and Prm3p colocalized with Kar5p in 79% of the shmoos (B). Localization of Prm3p to the vicinity of the SPB, the site of nuclear envelope fusion, is consistent with Prm3p's important role in karyogamy.
The colocalization of Prm3p with Kar5p in shmoos suggested that these proteins may be part of a protein complex that facilitates fusion of the nuclear envelope. To explore the interaction between Prm3p and Kar5p, we examined Prm3p localization in zygotes blocked at the initial stage of nuclear envelope fusion. GFP-Prm3p was enriched at the SPB in 96% (n = 151) of KAR5 zygotes (A). In the kar5Δ zygotes, the concentration of GFP-Prm3p at the SPB was reduced (B) or disappeared altogether in 29% (n = 96) (C). Measurement of the fraction of total GFP-Prm3p at the SPB (D) showed that the amount of GFP-Prm3p at the SPB was significantly decreased in the kar5Δ zygotes to 54% of wild-type levels (p = 7 × 10−52; Student's t test). In contrast, localization of Kar5p to the nuclear periphery was not affected by prm3Δ (data not shown). Furthermore, GFP-Prm3p localization in shmoos was not affected by mutations in KAR7, KAR8, SEC72, or PRM5. The increased localization of GFP-Prm3p at the SPB when Kar5p is overexpressed in shmoos, together with the decreased localization in kar5Δ zygotes, suggests that Kar5p is responsible for recruiting or stabilizing Prm3p to the vicinity of the SPB.
In a second approach to probing the interactions between Kar5p and Prm3p, we examined the genetic interactions between various mutations. Both prm3Δ and kar5Δ are “bilateral” mutations, requiring both parents to be mutant for nuclear fusion to be defective. When a prm3Δ cell was mated to a kar5Δ cell, a partial nuclear fusion defect resulted, resulting in 28% Kar− zygotes (). This “synthetic” bilateral defect was not observed when prm3Δ was mated to other kar mutants. A stronger defect was observed when prm3Δ was crossed to a partially dominant allele, kar5-1162, increasing to 72% (). Overexpression of Kar5p suppressed the prm3Δ nuclear fusion defect; nuclear fusion increased sixfold when KAR5 was overexpressed in one mating partner and 10-fold when KAR5 was overexpressed in both mating partners (). Overexpression of Prm3p did not suppress the mating defect in kar5Δ zygotes. Thus, both localization and genetic data suggest that Prm3p interacts with Kar5p.
PRM3 and KAR5 interact genetically
Prm3p Physically Interacts with Kar5p
To determine whether Prm3p interacts directly with Kar5p, we used coimmunoprecipitation (coIP) of proteins tagged with FLAG, HA, or GFP. Because coIPs of membrane proteins can be complicated by the harsh conditions required to extract the proteins, a membrane permeable cross-linker, dithiobis(succinimidyl propionate) (DSP) was used in some experiments. DSP is a cleavable cross-linker that reacts with the amine group on lysine residues (Walleczek et al., 1989
). Each experiment was performed with and without DSP; although coprecipitation was detected without the cross-linker, the efficiency was reduced.
When GFP-Prm3p and HA-Kar5p were coexpressed and anti-HA antibody was used to immunoprecipitate HA-Kar5p, a small fraction of the GFP-Prm3p coprecipitated (A), consistent with Kar5p's restricted localization near the SPB. As a control, the abundant 3XGFP-HDEL protein in the NE lumen did not coprecipitate with HA-Kar5p. The reciprocal experiment was performed with cells expressing FLAG-Prm3p and HA-Kar5p, by using anti-FLAG antibody to immunoprecipitate FLAG-Prm3p. HA-Kar5p was coprecipitated with FLAG-Prm3p (C), confirming the physical interaction between Prm3p and Kar5p.
Figure 9. Prm3p interacts with Kar5p. (A) Yeast protein extract containing HA-Kar5p and GFP-Prm3p (MS7590 with pMR2872 and pMR5062) were immunoprecipitated with anti-HA antibody. Total protein (column 1), unbound protein (column 2), and bound protein (column 3) (more ...)
The bulk of Kar5p resides within the NE lumen and only ~20 residues between the second and third transmembrane domains are predicted to be exposed to the cytosol (Beh et al., 1997
; Erdeniz and Rose, unpublished). Thus, the cytosolic loop is the only region of the protein that may be in contact with Prm3p. To determine the role of this region of Kar5p in interactions with Prm3p, coimmunoprecipitations were performed using cells expressing FLAG-Prm3p and HA-tagged kar5
deletion mutants (Erdeniz and Rose, unpublished). The kar5-hyd2
Δ mutation is a deletion of the second transmembrane domain, whereas the kar5-hyd3
Δ mutation is a deletion of the third transmembrane domain (B). The topologies of the kar5
deletion mutant proteins were determined using C-terminal His4C fusions (Sengstag, 2000
; Erdeniz and Rose, unpublished). In the kar5-hyd2
Δ mutant, the third transmembrane domain is oriented such that that the cytosolic loop is within the NE lumen; in the kar5-hyd3
Δ mutant, the loop remains cytosolic. The mutant proteins were expressed at levels comparable to the wild type (C). Comparison of coIPS of the kar5
mutants with the wild-type strain showed that the amount of Kar5p protein pulled down was significantly reduced in the kar5-hyd2
Δ mutant (10% wild type) but not in the kar5-hyd3
Δ mutant (90% wild type; C). Therefore, we conclude that the cytosolic loop of Kar5p is required for interaction with Prm3p, consistent with interaction on the cytoplasmic face of the nuclear envelope.