Localization of Fus2p-GFP in wild-type cells
encodes a 677-residue, 79-kD protein (), including a region (residues 116–327) that shares a homology with the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho-like GTPases (Rho-GEF, P = 1.9 × 10−35
, analyzed by SMART; Letunic et al., 2006
). A short motif (PTRRKYS) resembling an NLS is present within the Rho-GEF domain (residues 313–319). A region near the C terminus (538–574) is predicted to form a coiled coil (probability 0.8, analyzed by COILS; Lupas et al., 1991
). Bordering on the coiled-coil region (570–582) is a motif (FQNLQNQMKRELP) that is partially homologous to a highly conserved motif in Rvs161p. Homologues of FUS2
are found within closely related yeast species as well as more distantly related hemiascomycetes (e.g., Candida glabrata
, Debaryomyces hansenii
, Ashbya gossypii
, and Kluyveromyces lactis
; Dujon et al., 2004
Figure 1. Fus2p-GFP is functional and localizes to the shmoo tip. (A) FUS2 encodes a 79-kD protein with a Dbl Rho-GEF domain (DBH). A putative NLS is present at amino acid residues 313–319. A putative coiled-coil domain is present near the C terminus. (B) (more ...)
To identify the localization of functional Fus2p, we attempted to epitope tag both the N and C termini of the protein. In both cases, the resulting proteins were only partially functional (7 and 29% of wild-type levels of mating, respectively). Immunofluorescent microscopy showed two different patterns of localization in shmoos; the N-terminally tagged protein localized to the shmoo-tip and the C-terminally tagged protein localized to the nucleus (unpublished data). However, both proteins localized to the ZCF in prezygotes. Both termini are strongly conserved in other yeasts, which suggests that these regions are required for normal function. To identify functional sites to tag Fus2p, we compared the FUS2
sequence of closely related yeasts (Cliften et al., 2003
; Kellis et al., 2003
) to identify regions that were especially nonconserved and therefore likely to reside in nonessential surface loops. Two regions were chosen for internal epitope tagging (residues 104–109 and 410–419). In both cases, the FLAG-tagged proteins were fully functional (100% of wild-type mating efficiency) and localized to the shmoo tip and ZCF (unpublished data).
To observe Fus2p localization in live cells, we used the upstream internal site to insert GFP. Using a sensitive replica-plate mating assay to a fus1 fus2 partner, the Fus2p-GFP–tagged protein supported mating as well as either the Fus2p-FLAG construct or untagged wild-type Fus2p (). In quantitative matings with a fus1 fus2 partner, the Fus2p-FLAG construct supported mating as well as wild-type Fus2p, and Fus2p-GFP retained at least 90% of wild-type function ().
To observe the dynamic behavior of Fus2p-GFP, cells were first treated with α-factor to induce expression and allow cells to progress into the mating pathway. In cells that had arrested their cell cycle and formed shmoos, Fus2p-GFP exhibited three general types of localization (): diffuse in the nucleus, cytoplasmic dots, and cortical patches. Many of the cytoplasmic dots moved rapidly from the cell body to the shmoo tip, following roughly linear tracks at a mean velocity of 0.38 ± 0.19 μm/s (n = 135, maximal velocity = 0.99 μm/s). A few dots moved away from the shmoo tip and others exhibited random short-range movements. Cortical patches at or near the shmoo tip were very stable, showing no movement and no decrease in intensity over the course of the observation. Patches appearing at other cortical sites were not stable and disappeared over time.
To determine the order at which each localization pattern appeared, mitotic cells were treated with pheromone and observed by time-lapse microscopy at 10-min intervals (). In cells that had not completed mitosis, faint Fus2p-GFP fluorescence was observed in the nucleus (20–60 min). After cells completed mitosis, Fus2p-GFP was observed at the nascent shmoo tip (80 min). As the shmoo became more pronounced, Fus2p-GFP fluorescence at the tip increased, and cytoplasmic dots were observed (100 min). The later appearance of dots may reflect ectopic localization as cortical sites become saturated or the early low level of expression may have precluded detection. Regardless, these results show that Fus2p-GFP initially localizes to the nucleus and localizes to cytoplasmic sites after cells exit mitosis and enter the mating pathway.
Fus2p nuclear localization is regulated by the mating response
expression is pheromone regulated; in vegetative cells, FUS2
mRNA is virtually undetectable, increasing >32-fold after pheromone treatment (Roberts et al., 2000
). To eliminate expression effects on localization studies, FUS2-GFP
was placed under the control of the inducible GAL1
promoter. In vegetative cells in which PGAL-FUS2-GFP
was expressed by induction with galactose, Fus2p-GFP accumulated in the nucleus at all stages of the cell cycle (). When cells expressing PGAL-FUS2-GFP
were treated with α-factor, Fus2p-GFP stayed in the nucleus until the completion of mitosis and subsequently localized to the shmoo tip (). Thus, the export of Fus2p from the nucleus depends on signals from the pheromone response pathway, possibly in coordination with cell cycle control.
Figure 2. Fus2p localization is regulated by the mating response. (A) Mitotically expressed Fus2p-GFP localizes to the nucleus at all stages of mitotic growth. MY9201 containing PGAL-FUS2-GFP was grown in media containing galactose (top, DIC; bottom, GFP). (B) (more ...)
The apparent relocalization of Fus2p-GFP to the cortex could be caused by efflux from the nucleus or new synthesis of cytoplasmic protein coupled with turnover of nuclear protein. To distinguish between these two possibilities, vegetative PGAL-FUS2-GFP was induced with galactose and transcription was then repressed by the addition of glucose. The mRNA was allowed to decay (10, 20, and 30 min), after which α-factor was added to induce the mating pathway for 1.5 h (). For all periods of glucose repression, Fus2p-GFP localized to the shmoo tip and no Fus2p-GFP localized to the nucleus. The level of fluorescence at the shmoo tip was similar for all time points, indicating that residual FUS2-GFP mRNA did not make a substantial contribution to the signal. In cells where expression was maintained by continued presence of galactose, Fus2p-GFP stayed nuclear in the absence of pheromone. When pheromone was added to these cells, Fus2p-GFP localized to the shmoo tip and cytoplasmic dots. The signal at the shmoo tip was significantly stronger than when glucose was present, as expected for continued synthesis after pheromone addition. The results strongly suggest that nuclear Fus2p-GFP exits the nucleus and localizes to the shmoo tip in response to pheromone signaling.
Yeast cells exhibit a complex dose response to pheromone; 10–100-fold higher levels are required for shmoo formation compared with cell cycle arrest (Moore, 1983
). To determine whether Fus2p relocalization is simply a consequence of cell cycle arrest, cells containing FUS2-GFP
expressed from its normal promoter were treated with either standard (6 μM) or low (0.6 μM) levels of α-factor. At low pheromone levels, cells arrested but did not polarize to form shmoos, and low levels of Fus2p-GFP could be detected in the nucleus, confirming that pheromone induction had occurred. However, the Fus2p-GFP remained in the nucleus and did not localize to the cell cortex during the experiment (). In contrast, when cells were treated with high levels of pheromone, cells began to polarize soon after completing mitosis, and Fus2p-GFP localized to the tip of the shmoo and to cytoplasmic dots (). Thus, relocalization of Fus2p-GFP from the nucleus to the site of polarized growth requires high levels of pheromone beyond those sufficient for its transcriptional induction and cell cycle arrest.
The pheromone response signal is chiefly transmitted by activation of the MAP kinase Fus3p. The fus3
Δ mutants are defective for cell fusion, in part because of a defect in cell polarization (Matheos et al., 2004
). The fus3
Δ mutants respond transcriptionally to pheromone due to the presence of a second partially redundant MAP kinase, Kss1p. To distinguish the transcriptional response from other aspects of pheromone signaling, we examined a fus3
Δ mutant. Fus2p-GFP localized at the shmoo tip in 97% of wild-type cells treated with α-factor; it was retained in the nucleus in only 1% of cells. Fus2p-GFP was well-induced by pheromone in the KSS1+ fus3
Δ mutant but localized to the shmoo tip in only 2% of cells (). In >85% of cells, Fus2p-GFP was retained in the nucleus. In ~40% of the fus3
Δ cells, a small number of cytoplasmic dots were observed moving in the cytoplasm. These results show that Fus3p is specifically required for relocalization.
Rvs161p is required for Fus2p-GFP localization and movement
Fus2p interacts with Rvs161p during mating and Rvs161p is required for Fus2p's stability and function (Brizzio et al., 1998
; Gammie et al., 1998
). We therefore examined the localization of Fus2p-GFP in an rvs161
Δ mutant. In ~90% of rvs161
Δ shmoos, Fus2p-GFP failed to localize to the cortex or shmoo tip; most cells showed diffuse punctate localization in the cytoplasm (). The cytoplasmic dots did not undergo rapid transport; almost all showed short-range movements similar to Brownian motion. In some shmoos, faint nuclear localization was observed as well as a few dots close to the nucleus. In sum, Rvs161p is not required for Fus2p-GFP nuclear export but is required for Fus2p-GFP's rapid cytoplasmic movement and localization to the shmoo tip.
Figure 3. Rvs161p is required for Fus2p movement and localization. (A) Fus2p-GFP is not transported or localized in an rvs161Δ mutant. MY9214 was induced with pheromone for 1.5 h. (B) Fus2p-GFP localization is dependent on Rvs161p's cell fusion function. (more ...)
Rvs161p has two independent functions, endocytosis and cell fusion, and function-specific mutations have been isolated (end
). The fus
mutations are specifically defective for binding Fus2p (Brizzio et al., 1998
). To differentiate between defects in endocytosis or cell fusion, Fus2p-GFP was transformed into end
mutants. In an end-rvs161
mutant, Fus2p-GFP localized normally at the shmoo tip. In a fus-rvs161
mutant, Fus2p-GFP exhibited abnormal localization similar to the rvs161
Δ null mutant, neither localizing to the shmoo tip nor moving rapidly (). Therefore, Fus2p-GFP localization is dependent on the interaction with Rvs161p, and the mutant defect is not due to an indirect effect on endocytosis.
Rvs161p and Rvs167p form an obligate heterodimer during vegetative growth, and this complex is present after pheromone stimulation (Friesen et al., 2006
). To determine whether Rvs161p and Rvs167p are both associated with Fus2p in a ternary complex during mating, we performed coimmunoprecipitation (coIP) experiments in which all three proteins were detected (). In α-factor–treated cells, IP of either functional Fus2p-FLAG or Rvs167p-HA pulled down Rvs161p as expected (Brizzio et al., 1998
; Friesen et al., 2006
). However, in cells expressing both tagged proteins, IP of Rvs167p-HA was unable to pull down Fus2p-FLAG, and IP of Fus2p-FLAG was unable to pull down Rvs167-HA. We conclude that Fus2p and Rvs161p form a heterodimer during mating, separate from the Rvs161p/Rvs167p heterodimer, reflecting the different functions of Rvs161p in mating and endocytosis. When Fus2p-GFP was constitutively expressed under the control of the galactose promoter, formation of the Rvs161p–Fus2p complex was dependent on the pheromone response (), which is consistent with mitotic Fus2p being retained in the nucleus.
Fus2p transport and localization are not dependent on microtubules
The rapid linear movement of Fus2p-GFP dots suggested that they are actively transported along cytoskeletal filaments. Shmoos contain two different cytoskeletal elements along which movement might occur: (1) microtubules that extend from the nuclear envelope to the tip of the shmoo and (2) actin cables that form a network that emanates from the shmoo tip into the cytoplasm. To determine whether microtubules play a role in Fus2p localization, shmoos were treated with nocodazole to depolymerize microtubules (). In both mock-treated and nocodazole-treated cells, Fus2p-GFP localized normally to the shmoo tip and Fus2p-GFP dots moved rapidly through the cell (, arrows). Immunofluorescent staining for tubulin confirmed that the microtubules were depolymerized (). Thus, microtubules do not play a significant role in Fus2p localization or dynamics.
Figure 4. Fus2p movement and localization are independent of microtubules. MY9184 expressing Fus2-GFP expressed from its own promoter was treated with α-factor for 1.5 h and either mock treated (A and C) or treated with nocodazole (B and D) for 10 min. (more ...)
Fus2p localization is aberrant in polarity mutants
To determine if Fus2p-GFP localization is dependent on the actin cytoskeleton, we first examined mutants with defects in cell fusion due to defective actin organization: bni1
, and spa2. BNI1
encodes one of two formins required for actin cable nucleation (Evangelista et al., 1997
; Pruyne et al., 2002
; Sagot et al., 2002
). Bni1p is a substrate for the Fus3p MAP kinase and bni1
mutants fail to form polarized shmoos (Evangelista et al., 1997
; Matheos et al., 2004
mutants form peanut-shaped shmoos with broad shmoo tips due to disorganized actin cytoskeletons (Gehrung and Snyder, 1990
; Chenevert et al., 1994
; Valtz and Herskowitz, 1996
). Bni1p, Pea2p, and Spa2p are part of the polarisome complex, which helps orient the cytoskeleton toward the bud site in mitotic cells and the shmoo tip during mating (Sheu et al., 1998
Fus2p-GFP localized to the shmoo tip cortex in 97% of wild-type shmoos (). In bni1, pea2, and spa2 mutants, stable localization to defined regions of the cortex was observed in only 20–30% of cells. In most mutant cells, the Fus2p-GFP dots were randomly distributed. In cells where some dots were seen at the cortex, localization was unstable, disappearing after 10–20 min. We infer that the polarisome complex and/or proteins dependent on the polarisome are required for localization and maintenance of Fus2p-GFP at the cortex.
Figure 5. Fus2p localization and transport is dependent on actin organization. (A) Fus2p-GFP localization is disrupted in cell polarity mutants. Strains (MY9184 MY9208, MY9213, and MY9216) were treated with α-factor for 1.5 h (top, DIC or transmitted; bottom, (more ...)
Latrunculin disrupts Fus2p-GFP movement but not maintenance
To directly examine the requirement for actin, we used latrunculin-A (lat-A), which depolymerizes actin within a few minutes after addition. Because polarization requires an intact actin cytoskeleton, cells were first treated with α-factor to form shmoos and then treated with lat-A. In mock-treated cells, 94% of shmoos showed rapid movement of Fus2p-GFP dots (). After lat-A treatment, rapid movement was not seen in any cell (). In 11% of cells, we observed short-range movements, similar to Brownian motion. We conclude that rapid movement of the Fus2p-GFP dots is caused by actin-dependent transport.
In lat-A–treated cells, Fus2p-GFP localized to a broad region or several dots at the shmoo tip. To confirm that actin was depolymerized, cells were fixed and stained with Texas red–conjugated phalloidin. In control cells, cortical actin patches were concentrated at the shmoo tip (, top) and Fus2p-GFP colocalized with a subset of the patches. In lat-A–treated cells, phalloidin staining was diffuse or nonexistent (, bottom). Nevertheless, Fus2p-GFP remained at the shmoo tip, showing that actin is not uniquely required for the maintenance of cortical localization.
Cortical localization is dependent on both actin and Fus1p
We hypothesized that Fus2p-GFP remained localized to the shmoo tip in the absence of actin because it is anchored at the cortex by other proteins. The defects of the polarisome mutants implied that the putative cortical anchor might also be polarized. One candidate for a cortical anchor is Fus1p, a pheromone-regulated O-glycosylated integral membrane protein (Trueheart and Fink, 1989
). Fus1p localizes to a broad region at the shmoo tip (Trueheart et al., 1987
; Nelson et al., 2004
Deletion of FUS1 had surprisingly little effect on Fus2p-GFP localization. Cytoplasmic dots of Fus2p-GFP moved with wild-type velocity and concentrated at the shmoo tip (). However, actin-dependent transport might concentrate Fus2p-GFP at the shmoo tip and obscure an anchoring defect. Accordingly, we blocked actin-dependent processes in the fus1Δ mutant with lat-A. Strikingly, within 10 min after lat-A treatment, Fus2p-GFP became randomly distributed throughout the fus1Δ shmoos (). Texas red–phalloidin staining of the fixed cells confirmed that actin localization was normal in untreated fus1Δ cells and that lat-A treatment caused complete depolymerization ().
Figure 6. Cortical localization is dependent on both actin and Fus1p. (A and B) Fus2p-GFP localization is disrupted by lat-A in a fus1 mutant. MY9217 was treated with α-factor for 1.5 h and either mock treated or treated with lat-A as in . (A) The (more ...)
To quantify the effects of fus1Δ and lat-A on Fus2p-GFP localization, cell images were divided into 10 equal-width strips, orthogonal to the length of the cell. GFP fluorescence was measured in each strip and expressed as the fraction of total cell fluorescence (). In wild-type shmoos, almost half of the total fluorescence was found within the first 2/10 of the cell; the first 1/10 contained significantly more than the second 1/10 (28 vs. 18%). Remaining sections each contained 5–10% of the total in a decreasing gradient from the tip. The GFP fluorescence in untreated fus1Δ and lat-A–treated wild-type shmoos was very similar, albeit slightly less concentrated at the tip (fluorescence in the second 1/10 increased to 22 and 23%, respectively). In contrast, lat-A treatment of fus1Δ shmoos greatly reduced fluorescence at the tip (14%), and GFP became evenly distributed throughout all cell sections. Therefore, Fus1p and actin are jointly required for localization of Fus2p-GFP at the shmoo tip.
Localization of Fus2p in prezygotes and zygotes: an expanding ring during cell fusion
Localization at the shmoo tip positions Fus2p-GFP at the presumptive ZCF. In wild-type zygotes, Fus2p-GFP initially localized to a small patch at the ZCF (0.73 ± 0.17 μm, n = 11) that is considerably narrower than the overall width of the ZCF (, top). As cell fusion progressed, Fus2p-GFP expanded at a mean rate of 0.065 ± 0.02 μm/min along the ZCF to a diameter comparable to the junction between the cells (1.87 ± 0.31 μm, n = 20). Coincident with the expansion, Fus2p-GFP intensity at the ZCF progressively diminished, and increased localization was observed in the nucleus (, 30′–50′). In wild-type zygotes, 24 ± 5 min (n = 10) elapsed from the initiation of expansion to the final disappearance of Fus2p-GFP at the ZCF.
Figure 7. Fus2p-GFP localizes of to an expanding ring at the ZCF. (A) Mating of wild type (MY9184) expressing Fus2p-GFP to wild type (MY8093) for 1 h. Flattened projections of deconvolved images are shown (top). To reveal the ring, deconvolved images were rotated (more ...)
Because zygotes tend to orient parallel to the microscope slide, details of the expanding ZCF were obscured. To remedy this, images were captured at multiple focal planes during cell fusion and deconvolved image stacks were rotated about the x axis. From these images, it was evident that the initial patch of Fus2p-GFP developed into a ring, which then dilated as cell fusion progressed (, bottom).
To facilitate these studies, Fus2p-GFP cells were mated to fus1 fus2 mutants, which impeded cell fusion, making it easier to find and observe prezygotes. Cell fusion succeeded in ~30% of matings between wild-type and fus1 fus2 cells; a representative zygote is shown in . As in fully wild-type matings, Fus2p-GFP initially localized to a small region of the ZCF. Ring expansion began with a significantly larger patch of Fus2p-GFP (0.98 ± 0.33 μm, n = 17, P = 0.014), which expanded into a ring somewhat smaller than in the wild type (1.66 ± 0.42 μm, n = 19) before relocalizing to the nucleus. Ring dilation was significantly slower than in fully wild-type matings, occurring at less than half the rate (0.027 ± 0.009 μm/min, n = 16, P < 0.0001). The mean time between dilation initiation and Fus2p-GFP disappearance from the ZCF was significantly longer (36 ± 8 min, n = 8, P = 0.003). Therefore, in zygotes where functional Fus1p and Fus2p are supplied by only one parent, the overall rate of fusion is limited by their reduced concentration.
Cell fusion did not succeed in ~70% of wild-type–fus1 fus2
matings. In these prezygotes, Fus2p-GFP persisted at the ZCF for ~71 ± 17 min (n
= 29), forming a patch 1.20 ± 0.21 μm in diameter (n
= 32) before relocalizing to the nucleus (). During relocalization, Fus2p-GFP was frequently seen in cytoplasmic dots, possibly reflecting intracellular transport. Relocalization may reflect adaptation to pheromone signaling or reinitiation of polarization (Bidlingmaier and Snyder, 2004
). In some cases, frustrated prezygotic cells subsequently attempted to conjugate with another neighboring cell (unpublished data). In these cases, Fus2p-GFP relocalized to the ZCF associated with the new partner.
We next examined Fus2p-GFP localization in polarisome mutants (pea2, spa2, and bni1) mated to the fus1 fus2 strain (). As expected, very few such matings lead to productive cell fusion (<2%). Surprisingly, in >90% of these prezygotes, Fus2p-GFP did localize to the ZCF, albeit to significantly broader regions than the wild type (pea2, 1.78 ± 0.35 μm; spa2, 1.59 ± 0.13 μm; and bni1, 2.23 ± 0.48 μm; P < 0.0001). Like wild-type–fus1 fus2 prezygotes, at later time points, Fus2p-GFP increasingly localized to cytoplasmic dots and then relocalized to the nucleus (). However, Fus2p-GFP localization to the ZCF was significantly less persistent in the mutants than in wild-type cells (pea2, 50 ± 14 min; spa2, 52 ± 22 min; bni1, 48 ± 11 min; P < 0.0002), which suggests that actin organization contributes to the maintenance of Fus2p at the ZCF.
In fus1 mutants mated to fus1 fus2, Fus2p-GFP localized initially to a small patch at the ZCF (1.12 ± 0.29 μm), which is similar to the wild type. However, Fus2p-GFP very rapidly dispersed from the ZCF and relocalized to the nucleus significantly faster than wild-type or polarisome mutants (19 ± 5 min, n = 18, P < 0.0001), which is consistent with a role for Fus1p in retaining Fus2 at ZCF.
Fus2p-GFP was retained in the nucleus when fus3 mutant cells were mated with fus1 fus2 (, bottom). Unlike the polarisome mutants, Fus2p was not transiently localized at the ZCF but appeared nuclear at all time points. In ~30% of prezygotes, a small amount of Fus2p-GFP localized to the ZCF, indicating that the cells were not defective for localization per se. A defect in Fus2p export may contribute to the fus3 mutant cell fusion defect.