Binding of Boi1-PH to phospholipids
In all binding analyses reported in this study, we used Boi1-PH (see Fig. 1) rather than full-length Boi1, because we wanted to focus on interactions mediated by the PH domain. Also, we needed to have a soluble pool of protein for vesicle co-sedimentation studies, and while most of the Boi1-PH was soluble (at least when overexpressed), virtually none of the full-length Boi1 was soluble (whether or not overexpressed).
Fortuitously, we discovered that Boi1-PH can be proteolyzed in yeast extracts in a manner that is stimulated by PIP2. Figure 2A shows an example of this phenomenon; whereas most of the anti-Boi1 immunoreactivity was present in a single band when Boi1-PH was incubated with control PS vesicles that lacked PIP2, almost all of the anti-Boi1 immunoreactivity was present in faster-migrating bands when Boi1-PH was incubated with PS vesicles that contained PIP2 (lanes 1 and 5). (In this and all subsequent experiments that use PIP2/PS mixed vesicles, the mass ratio of PIP2:PS was 1:20.) Less total anti-Boi1 immunoreactivity was recovered when Boi1-PH was incubated with PIP2/PS mixed vesicles than when incubated with vesicles containing only PS (lanes 1 and 5), suggesting that the faster-migrating species represent degradation products of Boi1-PH. Consistent with this view, although Boi1-PH remained stable when incubated for longer intervals with vesicles that contained only PS, the anti-Boi1 immunoreactive bands became fainter and then disappeared altogether when incubated for longer intervals with PIP2/PS mixed vesicles (data not shown).
The finding that PIP2 can stimulate degradation of Boi1-PH suggests that PIP2 may bind to Boi1-PH in a manner that results in the exposure of a protease-sensitive site. We do not know the identity of the relevant protease. However, we found that the PIP2-stimulated proteolysis of Boi1-PH is strongly enhanced by Ca++ and is inhibited by EGTA (data not shown), suggesting that this protease may require Ca++ for activity.
Next, we asked whether proteolysis of Boi1-PH could also be stimulated by other inositol-based phospholipids. PI and PI3P (phosphatidylinositol-3-phosphate), presented in PS-based vesicles, appeared to stimulate slightly the proteolysis of Boi1-PH, but did so to a much lesser extent than did PIP2 (Fig. 2A, lanes 1–3 and 5). In contrast, PI4P (phosphatidylinositol-4-phosphate)/PS vesicles stimulated substantial proteolysis of Boi1-PH, although to a lesser extent than did PIP2/PS vesicles (Fig. 2A, lanes 4 and 5). These findings indicate that different phosphatidylinositides can stimulate proteolysis of Boi1-PH but differ greatly in their abilities to do so.
To investigate whether the nature of the bulk lipid in the vesicles affects the ability of phosphatidylinositides to stimulate proteolysis of Boi1-PH, we repeated this analysis using the neutral phospholipid PC (phosphatidylcholine) instead of PS. Using reaction conditions that were otherwise the same as those used when PS was the bulk lipid, no proteolysis of Boi1-PH was detected in the presence of any of the phosphatidylinositides except PIP2, which stimulated only a small amount of proteolysis (Fig. 2B). Thus, PS is much more effective than PC at facilitating the ability of phosphatidylinositides to stimulate proteolysis of Boi1-PH.
To investigate further whether Boi1-PH binds PIP2, we used a vesicle co-sedimentation assay. In this analysis, we asked whether an otherwise soluble fraction of Boi1-PH, after being incubated with PIP2-bearing vesicles, would now sediment in conditions (436,000 × g for 1 hr) that cause vesicles to pellet. (To inhibit the proteolysis of Boi1-PH, we performed this analysis in buffer that lacks Ca++ but that contains EGTA and two other protease inhibitors that were not present in the proteolysis assay.) First, we asked whether Boi1-PH could pellet with vesicles that lacked PIP2. We did not detect any pelleting of Boi1-PH with PC vesicles (data not shown), but found that Boi1-PH could pellet with vesicles composed of PS (Fig. 3, top panel), suggesting that Boi1-PH can bind PS.
Next, we asked whether the inclusion of PIP2 in the PS vesicles affected the ability of Boi1-PH to pellet. In the starting buffer conditions (phosphate-buffered saline without additional salt), a larger percentage of Boi1-PH pelleted when the vesicles contained PIP2 (Fig. 3, lanes 1 and 2). However, most of the Boi1-PH pelleted whether or not PIP2 was present. So, to try to accentuate the effects of PIP2 on Boi1-PH's co-sedimentation with vesicles, we repeated this analysis using buffer that contained additional concentrations of KCl. In the presence of 50, 100, and 200 mM additional KCl, the ratio of Boi1-PH that pelleted with respect to that which stayed in the supernatant was greater when using vesicles that contained PIP2 (Fig. 3, lanes 3–8), suggesting that Boi1-PH has a higher affinity for PIP2 than for PS.
In similar experiments using mixed PIP2/PC vesicles, all of the Boi1-PH stayed in the soluble fraction (data not shown), supporting the views that the composition of the lipid bilayer affects the ability of Boi1-PH to bind PIP2 and that PS promotes this association.
Mutant versions of Boi1-PH that are impaired in the ability to bind PIP2
To investigate whether binding to PIP2 may be important for Boi1 function, we sought to make mutant versions of Boi1-PH that are defective in the ability to bind PIP2. As a guide for designing such mutants, we used information about one of the PH domains from pleckstrin. Figure 4 shows those positions in the N-terminal PH domain of pleckstrin that have been implicated, based on NMR analysis, to contact PIP2[17]. Boi1's PH domain has identical or similar amino acids at most of the analogous positions (Fig. 4).
To obtain mutant versions of Boi1 that might be defective in the ability to bind PIP2, we created three sets of mutations that resulted in amino-acid subsitutions at some of those positions. We call these sets of substitutions "KK" (K785E and K786A), "TK" (T793A and K795E), and "KKTK" (K785E, K786A, T793A, and K795E; a combination of the KK and TK sets) (Fig. 4).
To investigate whether these substitutions affect the ability of Boi1-PH to bind PIP2, we first used the proteolysis assay. We found that, in conditions in which PIP2 stimulated proteolysis of most of the wild-type Boi1-PH, PIP2 did not stimulate the proteolysis of the TK, KK, and KKTK versions of Boi1-PH (Fig. 5A, lanes 3, 6, 9, and 12), suggesting that all three sets of substitutions may disrupt binding to PIP2 and/or PS.
To investigate further whether the three sets of substitutions affect the ability of Boi1-PH to bind these lipids, we used the vesicle co-sedimentation assay. In the presence of vesicles composed of only PS, an appreciable amount of Boi1-PH(KK) pelleted (Fig. 5B, second panel, lanes 3 and 4). However, the percentage of Boi1-PH(KK) that pelleted was less than that for wild-type Boi1-PH (Fig. 5B, top and second panels, lanes 3 and 4), suggesting that the KK substitutions reduce somewhat the binding affinity for PS. The TK substitutions had a more obvious effect: no Boi1-PH(TK) was seen to pellet with PS vesicles (Fig. 5B, third panel, lanes 3 and 4), suggesting that the TK substitutions impaired binding to PS to a greater extent than did the KK substitutions. As expected, given that the TK substitutions were sufficient to severely affect pelleting with PS vesicles, none of the Boi1-PH(KKTK) was seen to co-sediment with PS vesicles (Fig. 5B, bottom panel, lanes 3 and 4).
Whereas inclusion of PIP2 in the vesicles increased the amount of wild-type Boi1-PH that co-sedimented, PIP2 did not have an obvious effect on the co-sedimentation behavior of Boi1-PH(KK) (Fig. 5B, top and second panels, lanes 3–6), suggesting that the KK substitutions impair binding to PIP2. In contrast, the TK mutant version of Boi1-PH appeared to retain a slight ability to pellet with PIP2/PS vesicles (Fig. 5B, third panel, lanes 3–6).
None of the Boi1-PH(KKTK) was detected in the pellet fraction even when using vesicles that contained PIP2 (Fig. 5A, bottom panel, lanes 5 and 6), consistent with the view that the KKTK mutant is more severely impaired in the ability to bind PIP2 than are the KK and TK mutants.
Exploring the significance of binding to phospholipids
To investigate whether binding to phospholipid may be important for Boi1 function, we used a red/white, colony-sectoring assay to test whether the KK, TK, and KKTK mutant versions of Boi1 could substitute in function for wild-type BOI1 and BOI2. For this analysis, we used strain PY967, which lacks the genomic copies of BOI1 and BOI2 and which is kept alive by BOI1-bearing plasmid pPB799. pPB799 also contains the ADE3 color marker. Cells that have this plasmid are red, while those that fail to inherit it are white. PY967 cells form thoroughly red colonies (lacking white sectors), because cells that fail to inherit the plasmid lack BOI1 and so do not propagate. However, when transformed with a second plasmid that can substitute in function for BOI1, PY967 cells that fail to inherit pPB799 are still viable and so give rise to white sectors.
To test the different versions of BOI1 for function, we introduced them on plasmids into strain PY967, at both 23 and 30°C, and then scored the degree of sectoring of the resultant transformant colonies. For each plasmid tested, approximately 200 colonies from each transformation plate were analyzed at each temperature. As negative and positive controls for sectoring, we used an empty plasmid and one that contains wild-type BOI1, respectively. As indicated in Figure 6, the empty plasmid (-) gave no sectoring colonies, indicating that the level of background sectoring in this assay is low. The plasmid that contains wild-type BOI1 gave a range of sectoring abilities, with 71 and 79% of the colonies showing a moderate or heavy degree of sectoring at 23 and 30°C, respectively (Fig. 6).
At both temperatures, the TK mutant version of Boi1 allowed a substantial fraction of colonies to show either moderate or heavy sectoring (Fig. 6), suggesting that the TK mutant is able to provide whatever vital function normally is provided by Boi1/Boi2. However, this version of Boi1 appeared to function less efficiently than did wild-type Boi1: at 23°C, the percentage of colonies that showed either moderate or heavy sectoring with Boi1(TK) was only 29%, and, at 30°C, the percentage of colonies that showed moderate or heavy sectoring with Boi1(TK) was only 10%.
At each temperature, almost every one of the colonies of cells that contained either the KK or KKTK mutant versions of Boi1 showed no sectoring (Fig. 6), suggesting that these mutant versions of Boi1 provide little or no function.
To investigate whether the diminished amount of sectoring allowed by the different mutant versions of Boi1 might be due to effects of the amino-acid substitutions on the concentration of Boi1 (e.g., by decreasing the stability of Boi1), we used immunoblotting to compare the concentrations of the mutant proteins to that of wild-type Boi1. This analysis was done using a genomically boi1 BOI2 strain, so that the only version of Boi1 present was the one expressed from a plasmid. As shown in Figure 7A, each of the mutant versions of Boi1 is present at a concentration similar to or higher than that of wild-type Boi1, suggesting that the reduced functions of the mutant versions of Boi1 are not likely to be due to effects on their concentrations.
One potential role of the binding of Boi1 to phospholipid is simply to target Boi1 to the plasma membrane. If this were the only role for such binding, then attachment to a membrane-localization tag might be able to restore function to versions of Boi1 that are impaired in the ability to bind phospholipid. One type of membrane-localization tag is the myristoyl group, which is attached to the glycine residue of proteins, such as S. cerevisiae Gpa1 (the α subunit of the pheromone-responsive G protein) [18], that contain at their N-termini the sequence MGXXXS/T (where "X" can be any amino acid) [19]. Thus, we investigated whether fusion to the sequence MGCTVS, which is present at the N-terminus of Gpa1 and which we will henceforth refer to as "Myr", could restore function to the mutant versions of Boi1 that are impaired in the ability to bind phospholipid.
At the time that we initiated this analysis, the only lipid-binding-impaired version of Boi1 that we had generated so far was the KK mutant. In preliminary analyses, we found that Myr did not improve the ability of full-length Boi1(KK) to promote sectoring, but that it did improve the ability of the KK version of Boi1-PH to promote sectoring (data not shown). Therefore, in subsequent tests to ask whether Myr could restore function to mutant versions of Boi1 that are impaired in the ability to bind lipid, we used specifically the Boi1-PH segment of Boi1 rather than full-length Boi1.
First, we checked to see whether Myr had any inhibitory effects on the function of wild-type Boi1-PH. Myr-Boi1(PH) allowed a degree of sectoring similar to that for Boi1-PH without the tag (Fig. 6), suggesting that Myr does not impair the function of Boi1-PH.
Next, we asked whether Myr could improve the ability of the different mutant versions of Boi1-PH to function. At 23°C, 25% of colonies containing Boi1-PH(TK) without the tag showed moderate or heavy sectoring. This value rose to 81% when using the Myr-tagged version of Boi1-PH(TK) (Fig. 6), suggesting that Myr improves the ability of the TK mutant version of Boi1-PH to function. At 30°C, the effect of Myr was even more striking: without the tag, only 1% of the Boi1-PH(TK) colonies showed any degree of sectoring; however, with the tag, 90% of the colonies showed moderate or heavy sectoring (Fig. 6).
Myr also greatly improved the ability of the KK mutant version of Boi1-PH to cause sectoring: Myr-Boi1-PH(KK) allowed 59% moderate or heavy sectoring at 23°C and allowed 28% moderate or heavy sectoring at 30°C, compared to there being no sectoring at either temperature without Myr (Fig. 6). At 23°C, Myr also improved the function of the KKTK (the most severe) mutant version of Boi1-PH: at this temperature, whereas Boi1-PH(KKTK) without Myr did not allow any sectoring, Myr-Boi1-PH(KKTK) allowed 17% of the colonies to show moderate or heavy sectoring (Fig. 6). In constrast, when tested at 30°C, Myr did not affect significantly the amount of sectoring allowed by Boi1-PH(KKTK): 98% of the colonies of cells containing Myr-Boi1-PH(KKTK) were non-sectoring at this temperature (Fig. 6).
It is unlikely that the mechanism by which Myr improves the function of the different mutant versions of Boi1-PH is by increasing their stability, because the presence of Myr did not have an obvious effect on the relative concentrations of the different versions of Boi1-PH (Fig. 7B).
Localization of wild-type and mutant versions of Boi1 and Boi2
To investigate whether binding to phospholipid may be important for proper localization of Boi1, we used GFP (green fluorescent protein) fusions to identify first the patterns of localization of wild-type Boi1 and Boi2 and then those of the mutant versions of Boi1 that are impaired in the ability to bind phospholipid. The micrographs in Figure 8 show examples of the different patterns of localization observed for wild-type Boi1-GFP. In unbudded cells, Boi1-GFP was seen to be concentrated in a single spot at the cell cortex ("polar" pattern) in 70% of the cells (Fig. 8A). In small-budded cells, Boi1-GFP was concentrated in the bud in 89% of the cells (Fig. 8B). In cells containing large buds, Boi1-GFP was concentrated in the bud in 50% of the cells and was concentrated at the neck in 31% of the cells (Fig. 8C). Boi2-GFP showed distributions of localization patterns generally similar to those for Boi1-GFP (Fig. 8A,8B,8C). Thus, Boi1-GFP and Boi2-GFP both appear to be localized to sites of polarized growth throughout the cell cycle.
The KK, TK, and KKTK versions of Boi1-GFP all showed a polar pattern of localization in approximately half of the unbudded cells (Fig. 8A), suggesting that binding to phospholipid is not critical for this pattern of localization. In small-budded cells, the KK, TK, and KKTK mutant proteins showed localization to the bud in only 18, 21, and 27% of the cells, respectively (compared to in 89% of the cells for wild-type Boi1-GFP) (Fig. 8B). Thus, although the KK, TK, and KKTK mutant proteins all were able to localize to the bud in small-budded cells, they were localized and/or retained there less efficiently than was wild-type Boi1-GFP.
A more striking effect of the KK, TK, and KKTK substitutions on the localization of Boi1-GFP was seen in cells that contained large buds: Boi1-TK-GFP was concentrated in the bud in only 4% of large-budded cells, and Boi1-KK-GFP and Boi1-KKTK-GFP were concentrated in the bud in less than 1% of such cells (Fig. 8C). None of the sets of substitutions had an obvious effect on localization to the neck, however (Fig. 8C). These findings suggest that, in large-budded cells, binding to phospholipid may be important for the localization of Boi1 to the bud but not for localization to the neck.
Although our studies focus on roles of Boi1's PH domain, we were also curious to know which other portions of Boi1 contribute to its proper patterns of localization. In particular, we wished to know whether either the SH3 domain or the proline-rich (Bem1-binding) region of Boi1 is important for any pattern of localization. To address this issue, we used the following mutant versions of Boi1: the "S" mutant, which contains a Lys residue in place of a Trp residue at a highly conserved position in the SH3 domain; the "P" mutant, in which seven of the nine Pro residues in the Pro-rich region are replaced with Ala residues (a version of Boi1 that does not bind Bem1); and the "SP" mutant, which contains both the "S" and "P" changes [1].
The most striking effect of the SH3-domain mutation was on the localization of Boi1-GFP to necks: we never saw Boi1-S-GFP localized to necks (Fig. 8C), suggesting that the SH3 domain is required for such localization. In constrast, the SH3-domain mutation had no obvious effect on the localization of Boi1-GFP to the bud in either small- or large-budded cells (Figs. 8B and 8C). However, this mutation increased the frequency of localization to the periphery of large-budded cells, and it did so to a degree that approximated that to which it caused a decrease in the frequency of localization to the neck: in large-budded cells, whereas wild-type Boi1-GFP was localized to the periphery of the mother part of the cell in only 3% of the cells and to the neck in 31% of the cells, Boi1-S-GFP was localized to the periphery of the mother in 22% of the cells (and was never localized to the neck). These findings raise the possibility that failure to localize to the neck increases the likelihood that Boi1 will adopt or retain localization at the periphery of the mother part of the cell.
Another apparent effect of the SH3-domain mutation on the localization of Boi1-GFP was seen in unbudded cells, in which the percentage of cells that showed a polar pattern of localization for Boi1-S-GFP (24%) was lower than for wild-type Boi1-GFP (70%), and in which Boi1-S-GFP gave a higher percentage of cells that showed the diffuse pattern (62%) compared to that given by wild-type Boi1-GFP (24%) (Fig. 8A). These findings raise the possibility that one way in which Boi1-GFP acquires a polar distribution in unbudded cells involves prior localization to the neck, via the SH3 domain.
In all classes of cells, the distributions of localization patterns for Boi1-P-GFP were not notably different from those for wild-type Boi1-GFP, and the distributions of localization patterns for Boi1-S-P-GFP were not notably different from those for Boi1-S-GFP (Figs. 8A,8B,8C), suggesting that the proline-rich region is not important for any of the observed patterns of localization of Boi1-GFP.