Mapping of the Scp1 Binding Site on Actin
—Previous studies have demonstrated that binding sites for both drugs and proteins on actin can be mapped in vivo
by the use of mutant actin alleles (18
). 16 yeast strains, each expressing a different mutant allele of actin (in the absence of wild-type actin), were transformed with a GFP-Scp1 plasmid (4
). Localization of Scp1 was determined in each case. Two plasmids were used, one where Scp1 was overexpressed from a heterologous (galactose) promoter (5
) and one in which Scp1 was expressed from its own promoter (4
). In both cases the data are qualitatively similar, and only the data for expression from its own promoter are shown. shows the localization in wild type and a number of the mutant alleles. Of the 16 strains, 3 showed significantly reduced Scp1 localization to cortical actin patches (; ). To confirm whether the patches visible in these mutants were indeed associated with actin, we generated strains that also expressed the well characterized F-actin-binding protein Abp1 fused with mRFP. Localization of Abp1-mRFP and GFP-Scp1 was recorded simultaneously using a DualView beamsplitter using the same exposure time of 1 s. As shown in , Abp1-mRFP is clearly visible in cortical patches as is GFP-Scp1 in cells expressing wild-type actin. In each of the actin mutants Scp1 does co-localize to Abp1-containing spots but with a markedly reduced level of intensity. We also stained the three mutant alleles with reduced Scp1 localization with rhodamine phalloidin (supplemental Fig. 1). These data indicate that act1–102
strains have an actin staining pattern indistinguishable from that of actin, suggesting that the actin in these cells is fully capable of forming filaments and organizing these appropriately. Actin organization in act1–115
-expressing cells is more aberrant with no clear long cables and with non-polarized actin patches. This organization may suggest that actin in these cells is more dynamic and less well able to form stable structures such as cables.
FIG. 1. Localization of GFP-Scp1 in strains expressing different actin alleles. A, GFP-Scp1 was expressed in wild-type cells and 16 strains expressing different actin alleles (). Three of these, act1–102, act1–157, and act1–115 (more ...)
Localization of GFP-Scp1 in strains expressing different actin alleles
The position of the three allele mutations within the monomer is shown in and within a model filament in . The mutations were distributed across the monomer with, at first glance, no clear region to mark a site of interaction. However, one of the alleles that shows a reduced level of localization, act1–157
, actually resides in the ATP binding cleft (D157E), and we know this is unlikely to be accessible to binding. This mutant had been generated as part of a series of mutants that are affected in ATP binding dynamics, and in fact this mutant was demonstrated to have increased nucleotide exchange and filament turnover (21
). We postulated that as an actin bundling protein, Scp1, might bind preferentially to more stable actin, and if the levels of this are reduced in these cells due to increased actin turnover, this would explain the reduced actin association.
The mutations in act1–115
lies close to the actin-actin interface within the filament, and the two residues mutated (E195A and R196A) lie close to a loop that is believed to play an important role in stabilizing interfilament connections by forming a cross-filament bridge to a hydrophobic pocket (22
). Furthermore, residues immediately adjacent (Gly-197—Thr-203) participate in longitudinal interactions between protomers in the same strand via both hydrogen bonds and van der Waals forces (23
). Previously we have observed that despite this strain having relatively normal growth phenotypes, its actin is particularly susceptible to the effects of the actin monomer associating drug latrunculin-A, indicating that its actin is particularly dynamic or unstable (19
). As with act1–157
, we therefore postulate that the lack of binding in this case is due to the absence of sufficiently stable actin to allow Scp1 to bind rather than marking a bona fide
binding site. The remaining mutant, act1–102
, defined by mutations K359A and E361A, lies on the surface of the actin monomer in a region that is known to interact with a number of actin-binding proteins, and we, therefore, considered this as a likely binding sites for Scp1 on actin.
Because other actin binding proteins, Sac6 (yeast fimbrin) and Abp1, have been suggested to interact physically or genetically with Scp1, we also determined whether these proteins are able to localize in the act1–102
mutant. As shown in , both Sac6p and Abp1 localize in a wild-type pattern in this mutant. Thus, althoughAbp1 has been reported to interact with Scp1, the presence of Abp1 on actin is not sufficient to recruit Scp1. In addition, although Sac6p and Scp1 have an apparently additive role in stabilizing actin in the endocytic cortical patches, they do not use completely overlapping actin binding sites. This was further confirmed in the mutant act1–120
() in which Scp1 localizes as in wild-type cells but which is unable to localize Sac6 (20
FIG. 2. Localization of Abp1 and Sac6 occurs normally in the act1–102 strain. A, Abp1-GFP localizes to cortical actin patches in wild-type and in Δscp1 cells, demonstrating that Abp1 is not responsible for localizing Scp1 to actin in vivo. Bar (more ...)
In previous studies we have shown that overexpression of Scp1 is detrimental and prevents growth on appropriate growth media that causes SCP1
). We, therefore, reasoned that if Scp1 is unable to associate with actin in the act1–102
strain and if actin bundling is the cause of cell death in the overexpressing cells, then Scp1 overexpression might no longer be lethal in act1–102
cells. This was tested by transforming cells with a plasmid carrying SCP1
which overexpresses the gene only when cells are placed on media lacking methionine. As shown in , whereas both wild-type and act1–102
cells can grow in the presence of methionine, removal of this nutrient causes expression from the plasmid, and in the case of wild-type cells, this overexpression of SCP1
leads to cell death. On the other hand, act1–102
cells are able to grow in the presence of this higher level of Scp1, supporting the idea that the mutations in act1–102
have disrupted the binding interface with Scp1 and demonstrating that actin binding is the cause of cell death when Scp1 is overexpressed as previously postulated (9
FIG. 3. Overexpression of SCP1 is not lethal in the act1–102 strain. SCP1 was cloned behind a methionine promoter on a low copy number (centromeric) and high copy number (2μ) plasmid. Expression was induced when strains were plated on media lacking (more ...)
The importance of the residues mutated in act1–102
for Scp1 binding was then tested in vitro
. Actin was purified from yeast expressing wild-type or mutant act1–102
actin (as described under “Experimental Procedures”). Recombinant Scp1 was purified from bacteria as previously described (5
). Filamentous actin has been demonstrated to pellet at high g
, and therefore, co-sedimentation of a protein to F-actin is widely used to demonstrate actin binding (see “Experimental Procedures”). Different concentrations of Scp1 (0.5–6.0 μm
) were added to a constant concentration of F-actin (4.0 μm
). After incubation, samples were centrifuged at high g
. The resulting supernatants and pellets were run on gels, and these were analyzed using densitometry to determine the dissociation constant (Kd
) of binding of Scp1 to the wild-type and mutant actins. The Kd
of binding of Scp1 for act1–102
was calculated to be 7.9 ± 2.2 μm
, which is about 6-fold lower than the wild-type actin (1.3 μm
Mapping the Sites of Actin Interaction in Scp1
—In previous work we had demonstrated that Scp1 can bundle actin but itself was not a dimer (5
). This suggested that Scp1 might carry 2 actin-binding sites. A series of Scp1 truncation mutants were generated as plasmid borne GFP fusions. Plasmids carrying these mutants were transformed into yeast cells, and localization was observed. Actin organization was determined in cells using rhodamine-phalloidin staining. In all cases, a wild-type pattern of cortical actin patches and actin cables was observed (data not shown). shows the mutants generated within the known domain structure of Scp1. Scp1-(1–154) carrying only the CH domain did not show any detectable localization to cortical patches, suggesting it carries no actin binding sites as would be predicted for a single CH domain of this type (24
) (). The spots that were observed do not resemble the actin localization in these cells, which looks normal with multiple punctate spots (data not shown). This fragment had also previously been shown to not bind actin in vitro
). The mutants Scp1-(1–172) and Scp1-(1–180) do show some spots, although these are fainter, and there appears to be increased cytoplasmic staining (). This reduced staining of cortical patches may suggest that only one binding site may be present. This was tested in vitro
by generating Scp1-(1–180) and Scp1-(1–172). As shown in , Scp1-(1–180) has a Kd
of 7.1 ± 2.5 μm
compared with wild-type 2.1 ± 0.4 μm
. The Kd
for Scp1-(1–172), although clearly binding to actin, was too low to be calculated. Furthermore, in assays to measure actin bundling efficiency, actin bundling was not observed with the Scp1-(1–180) mutant (data not shown). In vivo
assays of Scp1 mutants described in the next section also demonstrate the likely position of actin binding sites. Taken together these data strongly indicate that one binding site for actin lies at its extreme C terminus between residues 173 and 200 in the calponin-like repeat. The fact that localization is observed with Scp1-(1–172) but not with Scp1-(1–154) indicates the second binding site lies in the region 154–172.
FIG. 4. Expression of GFP-Scp1 mutants to map actin binding sites in Scp1. A, schematic of Scp1 showing domain structure with a CH domain from residues 27–154 and a calponin like repeat (CLR) from 173 to 200. B, plasmid-borne GFP-Scp1 was mutated to generate (more ...)
A further point raised from other studies is the role of the putative Ser-185 phosphorylation site in Scp1. The mammalian homologue of Scp1, SM22, has been shown to be phosphorylated on the equivalent serine, Ser-181, and this phosphorylation reduces the actin binding capacity of the protein (25
). It has been demonstrated that for Scp1 an S185D mutant behaves relatively normally in vivo
, whereas the S185A mutant behaves more like a complete deletion (4
). We generated the GFP-Scp1 S185A and S185D mutants to determine whether the S185A mutant might have such a severe effect on function due to lack of localization to actin patches. However, as shown (), both mutants co-localize to actin patches marked with Sac6-RFP, indicating that at least one actin binding site is functional in these mutants. It also strongly indicates that it is the bundling activity rather than binding that is regulated through this site.
Formation of Cortical Patches Is Defective in Actin-bundling Mutants
—We and others have previously noted an overlapping function of the yeast fimbrin homologue Sac6 with Scp1 (4
). Deletion of scp1
alone has no discernible effects on actin organization, whereas deletion of sac6
alone has modest effects, and cells are still able to grow at broad range of temperatures. The combined deletion, however, has a severe growth phenotype, and cells are temperature-sensitive and have aberrant cortical actin structures (5
The questions we have asked are, Why does the cell express two actin bundling activities that both function within the cortical patch, and also, Does deletion of scp1 cause any detectable endocytic phenotype? GFP-Abp1 was used as a functional marker of actin patches forming during the endocytic process and was analyzed in wild type, Δsac6, Δscp1, and Δscp1Δsac6 cells. Kymographs showing the behavior of representative patches are shown in . The lifetime of the patches were also quantified (). Deletion of either sac6 or scp1 increases patch lifetime from 17.9 to 23.4 or 24.7 s, respectively. The double deletion showed an even more dramatic increase in lifetime to 35.8 s.
FIG. 5. The behavior of Abp1-GFP in wild-type and mutant cells. Abp1-GFP was expressed in wild-type cells and in mutants Δsac6, Δscp1, and Δsac6Δscp1. A, kymographs, 4 for each strain, depict behavior of patches over time at the (more ...)
We also addressed the in vivo function of the Scp1 mutants Scp1-(1–154) and Scp1-(1–172). We reasoned that if scp1 mutants have two actin binding sites, they should be able to bundle actin, and so cortical actin patches should behave in a similar way to wild type. If, however, bundling is defective and the mutants either do not bind or bind only one site, these patches should behave more akin to those in Δscp1 cells. Analysis of Abp1-GFP movement in cells expressing mutant Scp1 proteins was assessed. As shown () patches in these cells behave in a similar way to those in Δscp1 cells (lifetime: Scp1-(1–154), 25.5 ± 1.9 s; Scp1-(1–172), 23.2 ± 1.5 s), indicating that Scp1-(1–154) and Scp1-(1–172) are not functioning to bundle actin. However, because we see some cortical patch localization in Scp1-(1–172) (), we can surmise that one binding site remains functional.
We also investigated the paths taken by the invaginating vesicles and their rate of movement (). Initially, patches at the plasma membrane are relatively non-motile; there is then a slow inward movement that in wild-type cells occurs at 40 nm/s. This is followed by a fast movement of ≥80 nm/s. All stages of movement are impaired in the mutants, although the slow movement phase is not so greatly affected in the absence of scp1, suggesting that the most important role of Scp1 is at the later fast movement stage. Deletion of both bundling activities reduces the slow inward movement rate by 2-fold, whereas the fast movement stage is 5-fold slower. This demonstrates for the first time that bundling proteins play a key role not just at early stages of invagination but also at later stages, most likely after scission.
We then wanted to assess the proportion of the forming endocytic patches in the mutants that were able to enter the fast moving stage that usually occurs after about 250-nm movement from the membrane that is presumed to correspond to vesicles formed after scission (10
). This might be considered to indicate the proportion of vesicles that successfully form and enter the cell. As shown graphically in , whereas wild-type and Δscp1
cells both showed >70% of patches able to enter the fast movement stage, only 40% of patches in Δsac6
cells moved past 250 nm into the cell. The loss of both bundling activities severely compromised inward movement with less than 10% of patches moving over 250 nm into the cell. We then determined the effect of the deletions on endocytosis of the fluid phase marker Lucifer yellow. Mutants were incubated for 80 min with Lucifer yellow and then analyzed by fluorescence microscopy (). Staining of vacuoles can be clearly visualized for both wild type and the single mutants. Thus, although the Δsac6
cells show marked defects in movement of patches, the cells can still endocytose fluid phase markers sufficiently to be clearly visualized in this assay. In contrast, the Δsac6
cells show no obvious staining of the vacuole, reflecting the very low proportion of endocytic patches that were detected as moving into the cell.
FIG. 6. Deletion of bundling protein genes affects fluid phase endocytosis. A, movement beyond 250 nm corresponds to initiation of fast movement of patches in wild-type (wt) cells presumably because vesicle scission has occurred. The number of patches moving (more ...) Patches Containing Only Scp1 Are Distinct from Patches in Wild-type Cells
—Although our data show that Scp1 has a greater influence later in the endocytic process than Sac6, we also wanted to address the mechanistic differences between the cellular roles of Sac6 and Scp1. To investigate this we measured the intensity of forming Abp1-GFP patches. Abp1 is proposed to bind along the length of actin filaments (rather than at either end (27
)). Thus, the intensity of GFP-Abp1 is likely to indicate the accessibility of Abp1 binding sites within the patch. Intriguingly, the fluorescence intensity of cortical patches was significantly reduced in the Δsac6
cells even though these patches had a longer lifetime than those in wild-type cells. The intensity of the patches in the wild-type, Δscp1
, and Δsac6
strains was about equal (). These data indicate that in the absence of Sac6, there are fewer sites for Abp1 binding, resulting in lower intensity of patches. This change could be brought about if Scp1 partly functioned in place of Sac6 and if Scp1 interacted with actin in a different way to Sac6 to generate tighter bundles that are less accessible to Abp1 binding.
FIG. 7. Differences between Δsac6 and Δscp1 cells indicate different modes of interaction with actin. A, average cortical patch intensities were assessed for wild-type (wt) cells and in mutants Δsac6, Δscp1, and Δsac6Δ (more ...)
We also asked whether the reason that the single mutants had relatively minor phenotypes compared with the double deletion could in part be due to a compensatory increased level of one protein in the absence of the other. We made extracts of cells that were wild type or deleted for one or both proteins. These were then separated by SDS-PAGE and Western-blotted. The blots were probed with antibodies to Sac6 or Scp1. As shown in , deletion of sac6 causes an increase in the level of Scp1 in cells. This was quantified over three independent experiments, and data demonstrate an increase of about 50% in Scp1 levels in the absence of Sac6. The converse was also observed, with an increase in Sac6 protein of about 30% in cells that were deleted for scp1. Thus, cells appear to have mechanisms to compensate for the loss of bundling activity.
If the actin associated with the invaginating vesicle membrane is no longer tightly bundled, this might be expected to impact on the patch morphology. To address this, cortical patch diameters were measured as described under “Experimental Procedures” in the wild-type, Δscp1, Δsac6, and Δsac6Δscp1 strains. As shown in , whereas patches in wild-type and Δscp1 strains are very consistent in their diameter (208 and 210 nm, respectively), lack of sac6 causes the patches to increase in diameter to about 305 nm), and the lack of any actin-bundling activity produces a much larger size patch with a diameter almost double that of the wild-type cells (415 nm).
Scp1 Is Recruited to Cortical Patches Later Than Sac6—Our data in suggests that deletion of sac6 has an effect on the invagination stage of endocytosis, whereas scp1 deletion only affects the final, inward movement stage. Temporal control of endocytic complex assembly is likely to be important for ensuring that appropriate structures formed at the right time. To investigate the timing of recruitment of Scp1 and Sac6, we generated strains containing both Sac6 fused to an mRFP tag and Scp1 fused at the N or C terminus with GFP. Sac6-mRFP is considered to be a fully functional protein, as its expression in the absence of endogenous Sac6 behaves as a wild-type cell rather than exhibiting phenotypes associated with the SAC6 deletion. Scp1 fused to GFP at its N or C terminus generates a protein that co-localizes to actin patches. However, neither of the Scp1 fusions is fully functional, as the phenotype of a Δsac6 Scp1GFP strain is more compromised than the sac6 deletion alone (data not shown). Because the fusions localize appropriately, we still considered that meaningful data could be obtained by comparison of these fusions and that currently these approaches provide the best means to gain insights into spatiotemporal aspects of endocytic complex formation in cells. First, we measured the lifetime of all three fusions. As shown in , Sac6 lifetime is 16.15 ± 0.46 s. This is slightly shorter than that of Abp1 in our cells. Sac6 lifetime is, however, longer than that of Scp1. Interestingly, the lifetime of both N-terminal-tagged Scp1 (11.8 ± 0.8 s) is almost identical to that of the C-terminal-tagged protein (11.6 ± 1.0 s). Thus, the Scp1 fusion protein can localize correctly within the cell and has a lifetime appropriate for its incorporation into actin patches.
FIG. 8. Sac6 and Scp1 dynamics in endocytic patches. A, lifetimes of individual sac6-mRFP or GFP-Scp1 patches ± S.E. in wild-type and mutant cells. n > 37 patches for each strain. Movies were taken with 1-s frame intervals for both Sac6-mRFP and (more ...)
We then determined the relative time of recruitment for each protein. We have analyzed the N-terminal-tagged GFP-Scp1 because this fusion is furthest from the actin binding site and so potentially less likely to interfere with this function. By imaging the fusions proteins simultaneously, we could determine that Sac6 arrives at the forming endocytic patch 1.2 s before Scp1 and leaves 1.6 s after. Intensity profiles and distance plots were then generated for both Sac6-mRFP and GFP-Scp1 following four spots for each protein (). Co-localization of sac6RFP and GFP-Scp1 is shown in . These data show that Sac6 localizes to spots at the plasma membrane, reaching its greatest intensity just before movement away from the plasma membrane. GFP-Scp1 shows a broader distribution of intensities. On average, the greatest intensity is reached just at or after movement from the membrane has been initiated. This agrees with our data above that the lifetime of Scp1 is shorter and that it arrives after Sac6.