A New Strategy for Identifying Genes that Regulate Cell Fusion
We devised a strategy to identify mating-specific genes that may have escaped earlier genetic screens due to functional redundancy within or between mating partners. Such redundancy often produces a weak phenotype that can be difficult to detect. For example, in the case of the redundant genes FUS1
, a fus2
mutant displays little mating defect unless both mating partners also have deficiencies in FUS1
(Trueheart et al. 1987
). To avoid overlooking functionally redundant genes in our search, we employed a reverse genetic strategy that did not depend initially on the strength of the mutant phenotype. Specifically, we asked, “What pheromone-induced membrane proteins have not yet been studied?”
To address this question, we compiled already-published databases of gene expression data and gene properties, restructured them in a common format, and wrote a program to search this composite database. We used the program, called Webminer (see Materials and Methods), to examine gene expression profiles of cells arrested in G1 by treatment with the pheromone α factor. These genomic expression datasets were originally collected in the course of another group's study of cell-cycle transcription and made available online (Spellman et al. 1998
; http:// genome-www.stanford.edu/cellcycle). We reinterpreted the data to identify a number of strongly pheromone-induced proteins. As a second criterion, we demanded that potential target proteins have at least one hydrophobic domain, indicative of secretory or membrane proteins.
Specifically, we set an arbitrary cut-off to select genes that are induced more than threefold by mating pheromone. This criterion identified a set of 54 candidate open reading frames (ORFs) out of the 6,116 ORFs assayed in the genomic expression dataset (). We next assigned a score to every ORF to reflect its likelihood of encoding a membrane protein. To calculate these values, we wrote a program that scans predicted protein sequences in windows of 19 amino acid residues and assigns a hydrophobicity, or H, value to each window based on its amino acid composition. The hydrophobicity values we used are based on the empirically observed frequency of each amino acid's presence in known transmembrane domains (Boyd et al. 1998
). The highest H value among all of a protein's windows has been defined as that protein's MaxH (Boyd et al. 1998
). In most organisms, the MaxH values of all proteins fall into a bimodal distribution with a trough at 28.5 (Boyd et al. 1998
). Lower values represent the set of cytosolic proteins (e.g., Tub1p, α tubulin, has a MaxH of 22.5), and higher values represent membrane proteins (e.g., Hxt1p, a hexose transporter, has a MaxH of 30.9). In Saccharomyces cerevisiae
the bimodal distribution of MaxH values is present, but the overlap between the two sets is considerable. As a result, many known membrane proteins have MaxH values <28.5. We therefore set a less stringent threshold, by considering all ORFs with MaxH values >25 to be possible membrane proteins, yielding a set of 2,524 ORFs. This parameter narrowed our pool of 54 candidates to 20 genes, which we henceforth refer to as PRM genes (pheromone-regulated membrane proteins).
Figure 1 Identification of pheromone-induced putative membrane proteins by data mining. Dots represent the transcriptional induction in response to mating pheromone (y axis) and likelihood of coding for a membrane protein (x axis) of all 6,116 ORFs. ORFs induced (more ...)
Of these 20 genes, 10 have previously assigned functions (). Intriguingly, the identification of all 10 genes can be rationalized in light of roles they have in mating: four genes are involved in cell fusion (including the prototypical fusion genes FUS1
; Trueheart et al. 1987
) (, blue), three genes are involved in cell wall synthesis and remodeling (including AGA1
, which encode the mating agglutinins; Cappellaro et al. 1991
) (orange), and three genes are involved in other functions relevant to mating (including STE2
, which encodes the a
-specific pheromone receptor; Jenness et al. 1983
The remaining 10 ORFs had not been studied (; , red). Based on the successful identification of other membrane proteins involved in mating, they have a high likelihood of also being players in the process. We describe here the characterization of the most highly induced ORF, YNL279w, which we call PRM1.
Characteristics of the PRM Genes
Prm1p Is a Conserved Fungal Protein with Five Putative Transmembrane Domains
The predicted S. cerevisiae
Prm1p has clearly identifiable homologues in other fungi, such as C. albicans
(Contig 5-2425, position 7551–5680), S. pombe
(GenBank/EMBL/DDBJ No. 7630122), and Kluyveromyces lactis
(GenBank/EMBL/DDBJ No. AJ229977; Ozier-Kalogeropoulos et al. 1998
) ( A), but contains no recognizable motifs to hint at its function.
Comparison of Prm1p sequences from S. cerevisiae, C. albicans, and S. pombe. (A) Chemically similar, aligned amino acids are shaded. In the S. cerevisiae sequence, predicted transmembrane domains are overlined and potential glycosylation sites are boxed. (B) Schematic of proposed topology for Prm1p. All consensus glycosylation sites (S. cerevisiae) are marked with Y. The intensity of shading indicates the degree of sequence similarity between the three yeast homologues: the sequence is divided into 40 blocks, each 15 amino acids in length, and each block is shaded according to the number of conserved residues contained in a 45 amino acid window centered on it. Overall percent identity between sequences: S. cerevisiae and C. albicans, 20% identical; S. cerevisiae and S. pombe, 22% identical.
Prm1p has five conserved regions that, based on their hydrophobic character, are likely to span the membrane ( A, overlined). These putative transmembrane domains would divide the protein into two segments of ~175 residues each on one side of the membrane and two 50–100 amino acid segments on the other side of the membrane ( B). Together, both of the larger segments harbor 14 potential N
-glycosylation sites ( and , boxed and Y symbol), whereas the smaller ones have none. The large segments display the greatest sequence similarity between the three homologues, with about two thirds of the residues conserved. Intriguingly, these segments are identified as potential coiled-coil–forming regions by LearnCoil-VMF, a program designed to recognize viral fusases (Singh et al. 1999
). However, it is unlikely that a coiled-coil structure could assemble within a region of the protein that is anchored on both sides by transmembrane segments. The predicted overall picture of Prm1p, then, is that of a multispanning integral membrane protein presenting a large, evolutionarily conserved face on one side of the membrane and a smaller, less conserved face on the other ( B).
Pheromone Rapidly Activates Prm1p Expression in both Mating Types
To characterize Prm1p, we constructed strains carrying a fusion gene that appends an HA-epitope tag to the protein's COOH terminus (Prm1p-HA). We then assayed cells under mating or control regimes for the expression of Prm1p-HA by resolving total cell lysates with SDS-PAGE and visualizing Prm1p-HA by immunoblot.
Vegetatively growing cells did not express Prm1p-HA at detectable levels ( A, lane 1), but initiated expression within 5 min after addition of α factor (lane 2). After 20 min of pheromone treatment, the Prm1p level reached a maximum and persisted at steady state (lanes 5–7).
Figure 3 Expression profiles of Prm1p. (A, lanes 1–9) A strain of mating type a bearing a chromosomal copy of PRM1-HA (lanes 1–7 and 9), or a wild-type control strain (lane 8) was treated with 10 μg/ml alpha factor for 0–30 min, (more ...)
Western blot analysis identified Prm1p-HA (and by extension Prm1p) as several major forms: a sharp band migrating at 73 kD, the size predicted from the PRM1-HA open reading frame ( A, arrowhead), and a series of broad bands centered at roughly 115 kD (bracket). These species collapsed to a single band of ~73 kD after treatment with endoglycosidase H, indicating that the larger bands are heterogeneously glycosylated ( A, lane 9). The presence of extensive oligosaccharide addition confirms our prediction that Prm1p is initially integrated into the membrane of the endoplasmic reticulum and, based on the proposed topology in B, suggests that Prm1p may display its two large conserved segments on the lumenal or extracellular side of the membrane.
In addition to the newly synthesized and glycosylated forms, we also reproducibly observed a weaker band migrating at ~15 kD, which appeared after 30 min of pheromone treatment ( A, lane 7, *). Based on the position of the HA epitope, this band is likely to represent a COOH-terminal fragment, indicating that Prm1p-HA may undergo proteolytic processing during its maturation.
Cells of both mating types induce Prm1p when challenged with partners of the opposite mating type. Cells of mating type a expressed Prm1p-HA when mixed with untagged α cells for 30 min, but not when mixed with cells of the same mating type ( A, lanes 11 and 12). The converse is also true ( A, lanes 13 and 14): α cells expressed Prm1p-HA when mixed with untagged a cells, but not when mixed with untagged α cells. Prm1p-HA induction in α cells was weaker than in a cells, perhaps due to reduced diffusion of the lipophilic a factor compared with the more hydrophilic α factor.
The speed and extent of Prm1p expression during mating probably resulted from the presence of pheromone-responsive elements (PREs) upstream of the gene's coding sequence, as is true for many other mating-specific genes. The promoter of PRM1
contains three head-to-tail repeats closely matching the consensus PRE, TGTTTCAat
( B) (Yuan and Fields 1991
). The repeats are separated by a trinucleotide spacer TAC. These sequences appear 150–180 nucleotides upstream of the PRM1
coding sequence and probably serve as binding sites for the transcription factor Ste12p, a target of the MAP kinase cascade that links gene expression to the presence of extracellular pheromone (Herskowitz 1995
Prm1p Localizes to the Site of Cell Fusion
As a first step towards elucidating the function of Prm1p, we asked in what cellular compartment(s) the protein resides. To this end, we constructed strains bearing a chromosomal copy of a PRM1-GFP fusion gene driven by its own promoter, which allowed us to detect the Prm1p-GFP gene product by fluorescence microscopy.
Prm1p-GFP first became visible after 40 min of pheromone treatment as two rings, one encompassing the nucleus and one at the cell periphery ( A). This staining pattern is typical of the endoplasmic reticulum in yeast, consistent with Prm1p entering the secretory pathway.
Figure 4 Localization of Prm1p. (A–C) A strain of mating type a bearing a PRM1-GFP fusion gene was treated with 10 μg/ml α factor. Samples were taken and imaged on a confocal microscope after 40, 70, and 100 min of incubation, respectively. (more ...)
70 min after addition of α factor most cells have arrested in the G1 phase of the cell cycle, evidenced by their large unbudded state, and have begun to polarize. Prm1p accumulated in the “potbelly” formed by this polarization in addition to its persistent staining of the endoplasmic reticulum ( B).
By 100 min of pheromone treatment, most cells have formed mating projections, or shmoos. These shmoos would, in a more physiological setting, orient towards the greatest pheromone concentration and serve as the site where mating partners first make contact. Prm1p localized to the tip of the shmoo, where cell fusion would occur ( C).
We next mixed a and α cells, both bearing the PRM1-GFP fusion gene. In such physiological mating mixes, Prm1p-GFP localized at the midpoint of recently formed mating pairs, or zygotes, where two cells have met and initiated the steps required to degrade the intervening cell wall and fuse their plasma membranes ( D). In mating pairs that have already completed this fusion step, Prm1p-GFP formed a collar around the neck of the zygote ( E).
When the resulting diploid began to bud, Prm1p-GFP localized to the growing daughter ( F). Since diploids no longer express Prm1p (data not shown), the protein staining the first daughter was probably inherited from the parental cells.
More than Half of All Mating Pairs Deficient in PRM1 Fail to Fuse
To test whether Prm1p participates in cell fusion during mating as its expression profile and localization suggested, we constructed strains in which PRM1 was deleted by gene replacement (see Materials and Methods). When both mating partners lacked PRM1, we observed morphologically aberrant mating pairs by phase contrast microscopy. The most common aberration was the presence of a pronounced dark band at the mating pair neck, reflecting the undegraded cell wall between mating partners suggestive of a defect in cell fusion.
To monitor this phenotype more decisively, we constructed a Δprm1 α strain expressing a soluble, cytosolic form of GFP that marks its cytoplasm. This strain allowed us to readily distinguish fused zygotes from unfused mating pairs by scoring whether GFP had spread to both cells (indicating successful cell fusion) or remained restricted to one mating partner (indicating a failure to fuse). Using this assay, we observed unambiguously that matings between Δprm1 partners produced a mixture of fused zygotes and unfused mating pairs ( and ).
Figure 5 Δprm1 cells exhibit a fusion defect during mating. (A and B) Δprm1 a cells were mixed with Δprm1 α cells expressing soluble cytosolic GFP as a reporter of cytoplasmic mixing between mating partners. This mixture was applied (more ...)
We next quantitated the degree of the Δprm1 fusion defect using GFP-expressing wild-type and Δprm1 α strains. To do so, we mixed exponentially growing cultures of each of these strains with an appropriate partner strain, concentrated them on a filter, and placed the filter on a YPD plate where the cells were allowed to mate for 3 h. We then fixed the cultures for microscopy. At this point, zygotes produced by wild-type control cells were abundant but most were still freshly formed, having just begun to grow their first diploid bud.
In such mating mixes between wild-type control strains, 6% of zygotes/mating pairs scored as unfused ( C). Presumably, this baseline level reflects a kinetic intermediate in the mating reaction, and these cells would have eventually fused if the reaction were allowed to continue. Characteristically, these unfused mating pairs had a narrow neck. In contrast, when both mating partners lacked PRM1, 55% of zygotes/mating pairs were unfused, a ninefold increase over the number observed for wild-type strains ( C). These mating pairs may reflect either a kinetic delay in the fusion reaction, or they may represent a dead end in which some step in mating has gone awry and fusion cannot occur. At later time points the ratio of fused zygotes to unfused mating pairs did not appreciably change (data not shown), contrary to what a kinetic delay would predict. Moreover, in many of the mating pairs from a Δprm1 × Δprm1 mating, the neck diameter was significantly increased, indicating that these unfused mating pairs differed qualitatively from the ones observed at low frequency in the wild-type control reactions.
Is Prm1p required in both partners to promote efficient cell fusion? When one mating partner lacked PRM1 and the other was wild-type, we consistently observed a slight but significant fusion defect, with 12% of all mating pairs failing to fuse ( C). This defect was similar regardless of which partner carried the wild-type PRM1 allele. These results suggest that Prm1p functions symmetrically and can perform its duty even if present in only one mating partner, albeit at a consistently reduced efficiency.
Δprm1 Mating Pairs Form “Bubbles,” and Other Strange Shapes
In addition to the simple unfused phenotype shown in B typical of all fusion mutants, we observed more unusual morphologies in Δprm1 × Δprm1 matings. Notably, some mating pairs displayed intercellular bubbles, pockets of GFP-labeled or unlabeled cytoplasm from one mating partner that appeared to have invaded the other (, A–G). These bubbles appeared with approximately equal frequency in either direction: “innies” invading the α partner ( and ), and “outies” extending from the α cell into the a cell (, C–G). Bubbles varied in size and shape, ranging from tiny bulges in an otherwise straight cell–cell interface to large rounded pockets or, rarely, serpentine extensions that stretched across the entire length of the other mating partner.
Figure 6 The Δprm1 cells' failure to fuse sometimes results in intercellular bubbles. Mating mixes were prepared and imaged as described in . Representative images are shown. (A and B) “Innies” intruding from the a cell (nonfluorescent) (more ...)
Additionally, we observed one or both mating partners having budded a new daughter cell (, , and ). Budding indicates that a cell has escaped from the G1 arrest induced by exposure to mating pheromone and has reentered the cell cycle, committing itself to a new round of division. Apparently this release from pheromone arrest can occur even when surrounded by cells of the opposite mating type that are secreting pheromone and, in fact, even while adhered to one of them.
Lastly, some cells appeared to give up on the failed mating and, instead of budding, began to mate with another nearby partner. For instance, in H, the GFP-expressing cell in the bottom right seemed to have attempted to mate with the partner on the left and failed. It then went on to try anew with the cell on the right, while its original mating partner exited the mating arrest and began to bud.
The ability of these cells to exit G1 or to polarize towards a new partner and reinitiate mating suggests that Δprm1 mutants do not simply fuse more slowly than wild-type. Rather, the fact that they abandon their attempt at fusion indicates they have reached a dead end and would not form normal diploid zygotes even if given more time.
The Δprm1 Defect Results in Closely Apposed, Unfused Plasma Membranes
The bubbles suggested a breach of the cell wall between the mating partners, a phenotype unlike other fusion mutants. We used thin-section electron microscopy to examine this aspect of the Δprm1 defect more closely.
Many mating pairs exhibited an apparent dissolution of their cell wall at the center of the interface between the mating partners (, A–D). In most cases we found it necessary to examine serial sections through a single mating pair to find the point where a breakthrough occurred. The region of cell wall degradation almost invariably included the center of the cell–cell interface. In some cases it appeared restricted to the center ( and ), whereas in others it seemed to have spread asymmetrically to one edge of the mating pair (A and C).
Figure 7 Δprm1 cells successfully degrade their cell wall and juxtapose plasma membranes, but then fail to fuse. Mating mixes of Δprm1 partners were prepared as described above. The cells were then fixed, stained, and imaged by electron microscopy. (more ...)
Wild-type matings involve a similar local disruption of the cell wall at the center of this interface, followed by plasma membrane fusion and continued cell wall remodeling until the cytoplasmic bridge between the cells spans the entire width of the zygote and the cell wall becomes restricted to the periphery (Gammie et al. 1998
). Details of the intermediates after cell wall breakdown but preceding membrane fusion are unknown because they have not been captured by electron microscopy, presumably because these steps occur rapidly.
Δprm1 cells appeared to complete successfully the initial cell wall breakdown but then failed to perform plasma membrane fusion and continued cell wall remodeling. At the site where cell wall was removed in Δprm1 matings, the two plasma membranes came into close apposition (). Additional membrane appeared to be added to this region equally by both partners, generating bulges that are likely to correspond to the bubbles seen by fluorescence microscopy. Thus, the volume of one mating partner must have grown while the volume of the other one shrank by the same amount. Meanwhile their surface areas must have increased coordinately.
Vesicles of ~20-nm diameter were usually present in the bulge, often aligned in single-file rows oriented along a mating pair's long axis ( A), suggesting cytoskeletal attachment. These vesicles were packed with a densely staining material similar to that intervening between the two mating partners. These vesicles may deliver new membrane causing growth of the bulge.
Interestingly, the juxtaposed plasma membranes of the bulge were equidistant, consistently remaining separated by a gap of ~8 nm along their entire length (). A thin layer of densely staining material was seen between them, reminiscent of membrane adherence junctions found between mammalian cells.