Embed Size (px)
Citation preview
ACCELERATION OF YEAST ACTIN POLYMERIZATION BY YEAST ARP2/3 COMPLEX DOES NOT REQUIRE AN ARP2/3 ACTIVATING
PROTEIN#, !
Kuo-Kuang Wen and Peter A. Rubenstein From Department of Biochemistry, Roy A. and Lucille A. Carver College of Medicine,
University of Iowa, Iowa City, Iowa 52242 Running Title: NPF-Independent polymerization of yeast actin by yeast Arp2/3 complex.
# This work was presented at the 2004 meeting of the American Society of Cell Biology (Wen, K.K, and Rubenstein, P.A. (2004) Mol. Biol. Cell 15, suppl. 40a). ! Supported in part by a grant to PAR from the National Institutes of Health (GM33689) Address correspondence to: Peter A. Rubenstein. Department of Biochemistry, Roy A. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, Tel: 319-335-7911, Email: [email protected]
ABSTRACT
The Arp2/3 complex creates filament branches leading to an enhancement in the rate of actin polymerization. Work with Arp complexes from different sources indicated that it was inactive by itself, required an activating factor such as WASP, and might exhibit a preference for ATP or ADP-Pi actin. However, with yeast actin, Pi release is almost concurrent with polymerization eliminating the presence of an ADP-Pi cap. We thus investigated the ability of yeast Arp2/3 complex (yArp2/3) to facilitate yeast actin polymerization in the presence and absence of the Arp2/3-activating factor Las17p WA. yArp2/3 significantly accelerates yeast actin, but not muscle actin, polymerization in the absence of Las17p WA. Addition of Las17p WA further enhances yeast actin polymerization by yArp2/3 and allows the complex to now assist muscle actin polymerization. This actin isoform difference is not observed with bovine Arp2/3 complex, since the N-WASP VCA fragment is required for polymerization of both actins. Observation of individual branching filaments showed that Las17p WA increased the persistence of filament branches. Compared with WT actin, V159N mutant actin, proposed to be more ATP-like in behavior, exhibited an enhanced rate of polymerization in the presence of yArp2/3 complex. yArp2/3 caused a significant rate of
Pi release prior to observation of an increase in filament mass but while branched structures were present. Thus, yeast F-actin can serve as a primary yArp2/3 activating factor indicating that a newly formed yeast actin filament has a topology, unlike that of muscle actin, that is recognized specifically by yArp2/3.
INTRODUCTION
The proper functioning of the actin cytoskeleton
in processes such as cytokinesis (1), cell motility (2,3), endocytosis and exocytosis (4,5)and lamellipodial extension (6,7) requires that the system be dynamic both temporally and spatially. That is, the actin microfilament system must be capable of being rapidly assembled at a certain place in the cell at a certain time in response to a stimulatory signal and then be able to be disassembled once the particular task is completed. Recent works has demonstrated that an important factor in the nucleation and deposition of new filaments is the Arp2/3 complex (8).
This complex, found throughout evolution from yeast to mammals, is composed of seven protein subunits, two of which are the actin-related proteins Arp2 and Arp3 (9). Like actin, each is an adenine-nucleotide binding protein, and nucleotide hydrolysis is needed for proper complex function (10). Filament nucleation occurs by the complex preferentially binding to the side of a pre-formed filament creating a site for a branch of newly polymerized actin that extends from the mother filament at an angle of about 70o (11-14). Based on
1
JBC Papers in Press. Published on April 27, 2005 as Manuscript M502024200
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
studies carried out with Arp2/3 complex from mammalian and amoeboid cells (15), the complex alone appears to be in an inactive conformation which requires prior activation by another protein factor before filament nucleation can occur. A number of such activating factors, known as nucleation promoting factors (NPFs) have been identified, and one of the most well-documented is the WASP family of proteins. These proteins contain, at least, WASP homology 2 (W) and acidic (A) domains that are required for Arp2/3 activation and actin binding. In most cases, these elements are also in an inactive state until activated by interaction with an element of a signal transduction pathway. However, recent work with the WASP analogue from S. cerevisiae, Las17p, indicates that this family member may be constitutively active (16).
Initial experiments with the Arp2/3 system, using rabbit muscle actin, also indicated that the nucleotide state of the mother actin filament might be an important determinant in Arp2/3-dependent filament nucleation (17). With preformed filaments, Amann et al. observed that although branching occurred along the length of the filament, there seemed to be a preference for branch formation in the barbed end half of the filament (11). Ichetovkin et al (13) studied this nucleotide dependence in more detail. They showed that filaments formed with AMPPNP-actin seemed to have at least a two-fold higher propensity to form branches than those formed with either ADP or ADP-Pi actin, and they further demonstrated that branching was most effective with newly-formed F-actin. These studies led to the hypothesis that Arp2/3 complex has a preference for either the ATP- or ADP-Pi forms of F-actin or an immature, perhaps more stable form of the filament, seen predominantly at the barbed end of the mother filament (9).
In vivo studies involving the role of Arp2/3 utilize complexes eukaryotes ranging from mammalian non-muscle cells of various types to yeast. Yeast in particular has been a very valuable system because it has allowed a genetic dissection of the Arp2/3 complex and its interacting proteins (18-20). However, most in vitro studies involving the mechanism of Arp2/3-dependent enhancement of actin
polymerization have utilized muscle actin, in spite of the fact that a number of studies have shown distinct behavioral differences between yeast and muscle actins. Yeast actin polymerizes more rapidly than muscle actin (21,22), and EM studies have implied that the yeast actin filament is more open and flexible than its muscle actin counterpart (23). Yeast actin exchanges its bound adenine nucleotide much more rapidly than muscle actin, and ,more importantly, yeast actin releases its bound Pi following nucleotide hydrolysis much more rapidly than does muscle actin (24-27). At low actin concentrations, where most of the in vitro actin work is performed, a lengthy lag exists in muscle actin between the time the actin polymerizes and the time the Pi is released, whereas in yeast actin, under these same conditions, Pi release and polymerization are almost simultaneous. This difference is particularly significant in light of the discussion regarding the state of actin preferred by Arp2/3.
Because of the use of the yeast cytoskeleton as a model system for studying Arp2/3 function, coupled with the differences between yeast and muscle actins described above, we felt it was important to determine whether the particular actin used influenced the behavior of the Arp2/3 complex, and we also wished to compare the effectiveness of the mammalian and yeast Arp2/3 complexes with these two actins in the presence and absence of an NPF. In this paper, we report the results of experiments examining these parameters in terms of actin polymerization in solution, and we also examine them by looking at the effect of Arp2/3 on branching and debranching of individual actin filaments.
EXPERIMENTAL PROCEDURES
Protein Purification and Preparation--Yeast WT
and V159N actins were purified by DNaseI affinity chromatography as described by Cook et al. (28). Muscle actin was isolated from rabbit muscle acetone powder (29). A mixture of β and γ nonmuscle actins was obtained from Cytoskeleton, Inc., CO.
The actin was labeled on C374 with pyrene-maleimide (Sigma, MO) as described previously (30) and with a labeling efficiency between 80% and 95%. Actin was stored in G buffer (10 mM Tris-HCl, pH 7.5, 0.2 mM CaCl2, 0.1 mM ATP and 0.1 mM DTT) at 4°C and used within 3 days. ProtA-tagged yeast Arp2/3 was expressed and prepared
2
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
from the RLY1945 yeast strain (a generous gift from Dr. Li, R., Stowers Institute) as described by Pan, F. et al. (31) with modifications. Briefly, the cell lysate was prepared from the cells in early log phase by a glass-bead beater (Biospec, OK). ProtA-tagged yeast Arp2/3 in the cell lysate was affinity-purified by IgG Sepharose affinity chromatography (Amersham-Pharmacia), and digested with recombinant TEV to remove the ProtA tag. Finally, yeast Arp2/3 was further purified on a Source fast flow anionic exchange column (Amersham-Pharmacia). The preparations were analyzed by SDS-PAGE and visualized by Coomasie blue staining to assess the purity. They were then aliquoted, and stored at -80° C. Each aliquot was used within 2 days after thawing. GST-Las17pWA or GST-N-WASP was over-expressed in E. coli from plasmids provided by Bruce Goode, Brandies University, and Dr. Higgs, H., Dartmouth University, respectively. Both recombinant proteins were purified by glutathione affinity chromatography (Amersham-Pharmacia) as described (32) and further purified by Source fast flow anion exchange chromatography (Amersham-Pharmacia). The preparation quality was checked as described above for yeast Arp2/3. Bovine Arp2/3 protein complex was also provided by. Higgs.
Actin polymerization and Pi release--Actin polymerization in solutions containing different combinations of Arp2/3 complex and Las17p WA was triggered by the addition of MgCl2 and KCl to final concentrations of 2 mM and 50 mM respectively to produce F-buffer. Filament formation was monitored in a total volume of 120 µl. by the increase in light scattering with excitation and emission wavelengths at 360 nm as a function of time at 25 °C. The polymerization of pyrene-labeled (~5%) actin was also monitored by the change in pyrene fluorescence with excitation and emission wavelengths at 365 nm and 385 nm respectively. All experiments were recorded with a FluoroMax-3 (Spex, NJ). The release of Pi associated with actin polymerization was measured by the EnzChekTM phosphate assay kit (Molecular Probes, OR) (33) based on the manufacture’s suggestions.
Visualization of actin filaments with fluorescence microscopy--The procedure is modified from that of Boujemmaa-Paterski et al. (14). Yeast or muscle actin with yeast Arp2/3 in the presence or absence of Las17p WA was induced to polymerize as described above. Aliquots of the polymerization sample at different time points were incubated for 1 min with rhodamine phalloidin (Molecular Probes, OR) at a 1:1 mole ratio to actin. The sample was then diluted 20-100 fold into F buffer containing 0.1% 1,4-diazabicyclo[2.2.2]octan (DABCO, Sigma, MO), 10 mM DTT, and 0.1% methylcellulose. An aliquot of thee solution (1.6 µl) was applied to a poly-L-lysine (average M.W. 70 kDa at 2 µg/ml, Sigma, MO) coated glass cover slip, which is able to stabilize yet does not fragment filaments, and the sample was sandwiched onto a microscope slide. The sample slide was further sealed with nail polish to prevent evaporation. The filaments were observed with a Zeiss 200 M fluorescence microscope and a Hamamatsu ER ORCA camera. Branched and unbranched filaments were recorded and analyzed by the OPEN LAB software package (Imporvision, MA) and Image J (NIH, MD) respectively. At least 700 filaments were analyzed from each sample.
To monitor the location of branch formation on pre-formed yeast filaments, 4 µM pre-formed Alexa 488 labeled yeast actin filaments was stabilized by an equal concentration of phalloidin. This fluorescence labeled F-actin was fragmented by passage through a syringe with a 30G needle 5 times before use in experiments. Yeast G-actin, 5 µM, with 25 nM Arp2/3, and 0.5 µM Las17p WA was induced to polymerize as described above, following addition of an equal volume of 4 µM fragmented fluorescent F-actin. After 3 min., rhodamine phalloidin (Molecular Probes, OR) was added at a mole ratio at 1:3 with respect to G actin to stabilize actin filaments. The sample was diluted and monitored by the same fluorescence microscope assay as described above. Image processing was performed using Adobe Photoshop V 7.0 software.
RESULTS
Yeast actin polymerization in the presence of yArp2/3 and Las17p WA
To begin to characterize the biochemical behavior of the yeast actin/ yeast Arp2/3 complex (yArp2/3)
3
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
system, we assessed the relative ability of yArp2/3 alone to promote the polymerization of yeast vs. muscle actin. Figure 1 demonstrates a lack of ability of yArp2/3 to promote the polymerization of muscle actin, and it also depicts the faster polymerization of yeast vs. muscle actin in the absence of exogenous factors as has been shown previously. Surprising, however, was the significant enhancement of yeast actin polymerization by yArp2/3 in the absence of exogenous NPF. This result is contrary to the assumption that Arp2/3 requires a NPF in order to facilitate actin polymerization. A similar observation was recently published in a paper dealing with the effect of pan-1 on Arp2/3 function, although no comment was made on the differential effect of the muscle and yeast actins on yArp2/3 activity (19). To insure that the result with muscle actin was not due to a problem with either the actin or yArp2/3 preparation, we reassessed the ability of yArp2/3 to promote muscle actin polymerization in the presence of increasing amounts of the yeast WASP homologue, Las17p WA. Figure 2 demonstrates that increasing amounts of Las17p WA led to an increased ability of yArp2/3 to promote muscle actin polymerization. To further explore the apparent specificity of yArp2/3 complex for yeast actin, we repeated the experiments detailed above with a mixture of higher eukaryotic β and γ actins. Our results (data not shown) are identical with those we obtained with muscle actin: yArp2/3 alone showed no stimulation of polymerization but was active with this actin in the presence of N-WASP.
Since yArp2/3 alone could promote the polymerization of yeast actin, we wanted to determine if this acceleration of polymerization increased in the presence of Las17p WA. Figure 3A shows that increasing amounts of yArp2/3 lead to increasing degrees of acceleration of yeast actin polymerization, and Fig. 3B demonstrates that addition of Las17p WA enhances the ability of yArp2/3 to affect actin polymerization. A control reaction demonstrated that this polymerization was not due to the presence of Las17p WA alone.
Branch formation The enhanced ability of yArp2/3 to promote yeast
actin polymerization could arise for a number of reasons such as enhanced ability of the complex to bind to a mother filament, enhanced ability to nucleate a new filament by filament point end capping, or both. To discern the mechanism underlying this ability of yArp2/3 to function with yeast actin, we monitored the morphology of individual filaments at various times after the introduction of polymerizing salts to a solution of yeast G-actin and yArp2/3 complex. At the desired time, rhodamine phalloidin was added to an aliquot of the polymerizing solution, and the sample was visualized by fluorescence microscopy following its deposition on a glass slide. The results, shown in Fig. 4, reveal three different types of structures: single filaments, filaments with a single branch point, and filaments with multiple branch points regardless of whether yeast or muscle actin was used. With muscle actin, however, Las17p WA was included. With yeast actin, all three types of structures were observed whether or not the Las17p WA was included. The multiply-branched structures were similar to those reported by others (11,13) using muscle actin, bovine Arp2/3 complex (bArp2/3), and the N-WASP VCA domain.
The density of branches on the multiply-branched filaments varied over time: high branching density was observed predominantly during the nucleation phase. For example, the highly branched structure shown in Fig. 4e derives from a sample deposited on the slide within the first 30 sec. following initiation of polymerization. Medium density branching was predominant during the early elongation phase, and low density branching was present during the late elongation phase. Furthermore, in these reactions, the branches occurred randomly along the entire length of the filament. We were able to fit the decreasing portions of the curves in panels A-C to a first order equation. The analysis showed that the apparent first order rate constants for disappearance of branched yeast actin filaments were 0.25 min-1 and 0.10 min-1in the absence and presence of Las17p WA respectively. The rate constant for muscle actin in the presence of WA was 0.14 min-1, near that observed with the yeast actin under the same conditions. Overall, the presence of Las17p WA increased the persistence of branched structures in the sample.
4
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Previous works with bovine Arp2/3 complex (bArp2/3), muscle actin, and either ActA (14) or WASP (11,13) as a NPF showed that when pre-formed actin filaments were combined with the Arp2/3, branching was rare and, when it did occur, it was mainly observed in either filaments nucleated de novo or on those few filaments that had branched from a preformed filament. We thus assessed the ability of the yArp2/3 complex to polymerize yeast actin off of pre-formed F-actin in the presence of Las17p WA. Inclusion of the WA was to maximize the time of existence of multiply-branched filaments. Figure 6 shows that with the yeast proteins, too, branching from pre-formed filaments was rare and that most of the branching observed was either off of filaments initiated de novo or off of the barbed end of pre-formed filaments. Very few branches were observed attached to the middle of pre-formed filaments. It is possible that some of the discrimination between a branching preference for new vs. old actin results from the use of phalloidin-stabilized mature filaments since the phalloidin may have altered the topology of the yeast F-actin. Two observations argue against this view, however. Ichetovkin et al. (13) reported that phalloidin-stabilized filaments actually led to more efficient branching than filaments not stabilized with phalloidin, and Orlova et al. (34), based on EM reconstruction data, demonstrated that phalloidin F-actin produced a filament that was more like that caused by AMP-PNP actin rather than by ADP actin.
Effect of yArp2/3 and Las17p WA on Pi release from polymerizing actin
It had been previously established that during the polymerization of yeast actin at low concentrations, both hydrolysis of the bound ATP and subsequent release of Pi occurred concomitantly with polymerization. There was no lag in Pi release as is observed with muscle actin. We wished to know how Arp2/3-dependent branching of newly polymerized yeast actin affected Pi release relative to polymerization in the presence and absence of Las17p WA. The results are shown in Figure 7. Pi release can be divided into three stages relative to polymerization: nucleation, early elongation and steady state. Whether in the
absence or presence of Las17p WA, there is an initial burst of Pi release that precedes any detectable increase in light scattering that would signify polymerization. In the presence of Las17p WA, elongation and its concomitant Pi release occurs more rapidly than in the absence of Las17p WA, presumably reflecting Las17p WA -dependent stabilization of Arp2/3-actin complexes and related increased opportunity for subsequent actin polymerization. The continued rise in Pi release following completion of polymerization is due to monomer treadmilling in the filaments. The break in the Pi release curve between the treadmilling and polymerization phases occurs at a Pi amount equal to the amount of Pi expected to be released from a single turnover of each polymerizing monomer considering the actin concentration used and the critical concentration for the protein. This result would seem to preclude pre-polymerization monomer cycling as a major cause of the initial increase in Pi observed prior to detection of an increase in filament mass.
Effect of the V159N actin mutation on yArp2/3-dependent F-actin formation
The V159N mutation in yeast actin has been reported to produce a hyper stable filament (35), and subsequent EM characterization of these filaments show them to be in more like an ADP-Pi conformation than an ADP-conformation (23). If Arp2/3 complex had an enhanced affinity for ATP actin, ADP-Pi actin (11,13,14,17) or immature ADP-actin topology over mature ADP-actin filaments, there might be an increased efficiency of branching with this mutant actin in the presence of yArp2/3. Figure 8 shows that although V159N and WT actin polymerize approximately at the same rate under the conditions of our experiment, the introduction of yArp2/3 substantially accelerates its rate of polymerization compared to that observed with WT actin. Observation of individual filaments formed with V159N actin revealed the same range of branch morphologies we observed with WT and muscle actins.
We next assessed the effect of the actin mutation on branch persistence as shown in Figure 9. Analysis of the curve yielded a first order rate constant 0.09 min-1, approximately the same as obtained with WT actin in the presence of Las17p WA. These results correlate with the enhanced
5
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
filament stability of V159N actin reported previously.
Bovine Arp2/3 complex enhances yeast actin polymerization only in the presence of activated N-WASP
Rodal et al. (36) showed that Arp2/3 complex could exist in three identifiable conformations: an open, an intermediate or closed conformation, and that there was approximately twice as much of the yeast complex in the closed conformation as there was bovine complex. We demonstrated above that yeast actin was much better able to be polymerized in the presence of yArp2/3 than was muscle actin. This result may have been due to an enhanced ability of yeast actin to pull the Arp2/3 complex into the more closed, active conformation. If our hypothesis was correct, yeast WT and V159N actins might be more effective than muscle actin in leading to an activated form of bArp2/3 as well. Fig. 10, however, shows that this was not the case. The only way in which bArp2/3 promoted polymerization of yeast actin was in the presence of an activated form of N-WASP.
DISCUSSION
Despite the apparent degree of conservation
among Arp2/3 complexes from different organisms, the behavioral differences we and others (21,22,26,27,37) have documented in yeast actin compared with muscle actin suggested the possibility that there was co-evolution of these two proteins to maximize the efficiency of some physiological function in yeast. The focus of this paper was to test such a possibility. Our results show for the first time that, with yeast actin, yArp2/3 is capable of substantially promoting actin polymerization in the absence of a NFP. This characteristic derives from the differential ability of yeast actin vs. muscle or higher eukaryotic nonmuscle actins to interact with the complex since, under the same conditions, yArp2/3 required an activating protein to promote muscle and nonmuscle actin polymerization. Although yeast actin polymerization could be promoted by yArp2/3 alone, even greater rates of polymerization were obtained in the presence of Las17p WA. This difference between yeast and
muscle or the nonmuscle actins does not translate to other Arp2/3 complexes, since none of the actins could activate bovine Arp2/3 complex unless N-WASP was present.
Our results suggested that a yArp2/3-specific conformation facilitates the preferred interaction with yeast compared to muscle actin. X-ray crystallography demonstrated that the Arp2/3 complex exists in an open form which the authors then hypothesized closes upon activation, thereby allowing the Arp2 and Arp3 subunits to interact (38,39). This model was supported by work from Goley et al. (40) who used FRET to document closure of the complex upon activation by an activator protein. Based on the EM analysis of Rodal et al., as stated above, the ratio of closed states for yeast vs. bovine Arp2/3 complex is 2:1. If one groups the intermediate and closed states together, this ratio for yeast vs. bovine Arp2/3 complex is 4:1. These structural differences indicate a greater propensity of yArp2/3 for a conformation that would lead to its activation. However, in neither case is the ratio large enough to account for the all-or-none behavior we observed with yeast vs. muscle actin in the presence of yArp2/3.
Since the major function of the Arp2/3 complex apparently is to facilitate localized actin polymerization by initiating branches, assessment of bulk actin polymerization in solution is not the optimal way to assess Arp2/3 function. To form a filament branch, the Arp2/3 complex must interact with actin in two different ways. It must bind to the side of a pre-existing filament, and the Arp2 and 3 subunits must be in a conformation to allow nucleation and filament elongation of the branch. Either means of interaction represents a potential source of Arp2/3 activation brought about by yeast actin. The filament topology specific to yeast F-actin, following attachment of the complex, might lead to the closed activated state needed for new filament nucleation. Another origin of the difference could be caused by something as simple as a difference in surface charged residues between the yeast and no-yeast actins. We cannot discern between these possibilities. In other words, yeast F-actin is functioning as a NPF. A similar role for F-actin, that of a secondary activator of Arp2/3 complex, had been proposed previously by Volkmann et al. (41) on the basis of their EM reconstruction work.
6
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Alternatively, differences in behavior of the yeast vs. muscle actin monomers could promote formation of an intermediate conformation of the complex that would facilitate side-binding to an actin filament. This interaction would then promote final closing of a complex already prone to closure. We cannot differentiate between these possibilities at this time. What does seem apparent from our results is that the ability of yeast actin to work alone with yArp2/3 does not stem from its ability to cause the complex to initiate filament formation de novo since isolated filaments are not observed to any extent during the early phases of the polymerization process.
The additional activation of Arp2/3-dependent branching we observed in the presence of Las17p WA might result in part from more efficient closure of the complex to the active state and, in part, stabilization of the branched complex once it forms. Visualization of individual filaments formed by yeast actin and yArp2/3 in the presence or absence of Las17p showed virtually the same branching behavior. In the presence of Las17p WA, however, the lifetime of the branched structures appeared to be lengthened; that is, Las17p appeared to stabilize the branch junction formed by Arp2/3 with the mother filament. This stabilization would result in a more persistent presence of a pointed-end filament cap which could translate into the increased rate of polymerization we observed in solution assays in the presence of Las17p WA. It is unlikely that the Las17p is working by affecting the nucleotide state of the filament since, as we have shown previously with yeast actin, Pi release is almost concomitant with polymerization and ATP hydrolysis (27). Interestingly, when we repeated the experiment in the presence of pre-formed F-actin, branches from the middle of old filaments were rare. This result is similar to that observed by others with Arp2/3 complexes from different sources (11,13,14).
This difference in branching preference for new vs. pre-formed filaments has led to the proposition that Arp2/3 complex might have a preference for binding to ATP or ADP-Pi F-actin formed during the initial stages of polymerization rather than the mature ADP-F-actin conformation that characterizes mature
filaments as discussed above. Our previous work showing the absence of such a lag in Pi release under these conditions (27), would seem to eliminate this possibility. We have recently demonstrated using yeast actin that, subsequent to Pi release, there is a slow change to the mature ADP-F-actin conformation based on the change in intrinsic tryptophan fluorescence of the protein, and it may be that this immature ADP* F-actin conformation is what is being recognized by the yeast Arp2/3 complex (37).
Belmont et al. (23) had originally proposed that the hyperstability of the V159N mutant yeast actin they constructed resulted from the F-actin remaining in a more stable ATP-like conformation, even though during the polymerization of the protein, ATP hydrolysis and Pi release were normal. Subsequently we showed that, at least in terms of subdomain 1 of actin, this mutant underwent the same post Pi-release conformational change as did WT yeast actin (37). However, during the initial stages of polymerization it is possible that the mutant filament remained in a more ATP-like conformation that might be reflected by enhanced Arp2/3-dependent actin polymerization compared with that observed with WT actin. Our results are consistent with this hypothesis, even though polymerization rates for the two actins in the absence of Arp2/3 complex are essentially the same.
Finally, our assessment of Pi release kinetics during Arp2/3-dependent yeast actin polymerization led to a surprising observation. There was an Arp2/3-dependent stimulation of Pi release prior to our detection of filament formation by an increase in light scattering. The observation is perplexing in that during this time period, small densely branched structures (Fig. 4e) are observable microscopically. It is possible that they are too small to produce a detectable increase in light scattering in the concentration at which they are present.
Our results show that an accurate depiction of Arp2/3 function should be carried out with homologous proteins in order to obtain a behavior that might reflect how this system works in vivo. The ability to make mutant yeast actins and mutant Arp2/3 complex (20,31,36) subunits in yeast presents a way that one might use to delineate the mechanism by which yeast actin exerts this specific effect on the yeast Arp2/3 complex.
The ability of yeast Arp2/3 alone to efficiently nucleate yeast actin filament formation in vitro leads
7
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
to the question of the biological significance of this observation. First, actin in yeast is present only at a concentration of about 2 µM, significantly less that what is found in a higher eukaryotic cell (42). Yet, cytoskeletal function has to be spatially and temporally very efficient since the cell divides about every two hours. Perhaps the ability of Arp2/3 alone to facilitate yeast actin polymerization arose in part to meet the efficiency needed for maximal cell function. Our results demonstrate that Las17p can make this process generally even more efficient. However, a major part of is function may be in
spatial regulation of actin polymerization by its ability to recruit actin polymerization to a specific part of the cell. Deletion of this protein leads to a severe disruption in the ability of the cell to form actin patches in the growing bud where this protein normally localizes (43).
ACKNOWLEDGEMENTS
We would like to thank Bruce Goode, Brandeis
University, Harry Higgs, Dartmouth University, and Rong Li, Stowers Institute for their generosity in providing reagents used in this work.
8
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
References
1. Pelham, R. J., and Chang, F. (2002) Nature 419, 82-86 2. Lauffenburger, D. A., and Horwitz, A. F. (1996) Cell 84, 359-369 3. Mitchison, T. J., and Cramer, L. P. (1996) Cell 84, 371-379 4. Eitzen, G. (2003) Biochim Biophys Acta 1641, 175-181 5. Engqvist-Goldstein, A. E., and Drubin, D. G. (2003) Annu Rev Cell Dev Biol 19, 287-332 6. Svitkina, T. M., and Borisy, G. G. (1999) J Cell Biol 145, 1009-1026 7. Bailly, M., Ichetovkin, I., Grant, W., Zebda, N., Machesky, L. M., Segall, J. E., and
Condeelis, J. (2001) Curr Biol 11, 620-625 8. Pollard, T. D., and Borisy, G. G. (2003) Cell 112, 453-465 9. Pollard, T. D., and Beltzner, C. C. (2002) Curr Opin Struct Biol 12, 768-774 10. Dayel, M. J., Holleran, E. A., and Mullins, R. D. (2001) Proc Natl Acad Sci U S A 98,
14871-14876 11. Amann, K. J., and Pollard, T. D. (2001) Proc Natl Acad Sci U S A 98, 15009-15013 12. Mullins, R. D., Stafford, W. F., and Pollard, T. D. (1997) J Cell Biol 136, 331-343 13. Ichetovkin, I., Grant, W., and Condeelis, J. (2002) Curr Biol 12, 79-84 14. Boujemaa-Paterski, R., Gouin, E., Hansen, G., Samarin, S., Le Clainche, C., Didry, D.,
Dehoux, P., Cossart, P., Kocks, C., Carlier, M. F., and Pantaloni, D. (2001) Biochemistry 40, 11390-11404
15. Welch, M. D., and Mullins, R. D. (2002) Annu Rev Cell Dev Biol 18, 247-288 16. Rodal, A. A., Manning, A. L., Goode, B. L., and Drubin, D. G. (2003) Curr Biol 13,
1000-1008 17. Blanchoin, L., Pollard, T. D., and Mullins, R. D. (2000) Curr Biol 10, 1273-1282 18. Goode, B. L., Wong, J. J., Butty, A. C., Peter, M., McCormack, A. L., Yates, J. R.,
Drubin, D. G., and Barnes, G. (1999) J Cell Biol 144, 83-98 19. Toshima, J., Toshima, J. Y., Martin, A. C., and Drubin, D. G. (2005) Nat Cell Biol 20. Martin, A. C., Xu, X. P., Rouiller, I., Kaksonen, M., Sun, Y., Belmont, L., Volkmann, N.,
Hanein, D., Welch, M., and Drubin, D. G. (2005) J Cell Biol 168, 315-328 21. Buzan, J. M., and Frieden, C. (1996) Proc Natl Acad Sci U S A 93, 91-95 22. Kim, E., Miller, C. J., and Reisler, E. (1996) Biochemistry 35, 16566-16572 23. Belmont, L. D., Orlova, A., Drubin, D. G., and Egelman, E. H. (1999) Proc Natl Acad Sci
U S A 96, 29-34 24. Carlier, M. T., Dardick, I., Lagace, A. F., and Sreeram, V. (1986) Int J Gynecol Pathol 5,
69-74 25. Melki, R., Fievez, S., and Carlier, M. F. (1996) Biochemistry 35, 12038-12045 26. Yao, X., Grade, S., Wriggers, W., and Rubenstein, P. A. (1999) J Biol Chem 274, 37443-
37449 27. Yao, X., and Rubenstein, P. A. (2001) J Biol Chem 276, 25598-25604. 28. Cook, R. K., Blake, W. T., and Rubenstein, P. A. (1992) J Biol Chem 267, 9430-9436 29. Spudich, J. A., and Watt, S. (1971) J Biol Chem 246, 4866-4871 30. Feng, L., Kim, E., Lee, W. L., Miller, C. J., Kuang, B., Reisler, E., and Rubenstein, P. A.
(1997) J Biol Chem 272, 16829-16837 31. Pan, F., Egile, C., Lipkin, T., and Li, R. (2004) J Biol Chem 279, 54629-54636 32. Winter, D., Lechler, T., and Li, R. (1999) Curr Biol 9, 501-504 33. Webb, M. R. (1992) Proc Natl Acad Sci U S A 89, 4884-4887
9
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
34. Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J Mol Biol 245, 598-607 35. Belmont, L. D., and Drubin, D. G. (1998) J Cell Biol 142, 1289-1299 36. Rodal, A. A., Sokolova, O., Robins, D. B., Daugherty, K. M., Hippenmeyer, S., Riezman,
H., Grigorieff, N., and Goode, B. L. (2005) Nat Struct Mol Biol 12, 26-31 37. Bryan, K. E., and Rubenstein, P. A. (2005) J Biol Chem 280, 1696-1703 38. Nolen, B. J., Littlefield, R. S., and Pollard, T. D. (2004) Proc Natl Acad Sci U S A 101,
15627-15632 39. Robinson, R. C., Turbedsky, K., Kaiser, D. A., Marchand, J. B., Higgs, H. N., Choe, S.,
and Pollard, T. D. (2001) Science 294, 1679-1684 40. Goley, E. D., Rodenbusch, S. E., Martin, A. C., and Welch, M. D. (2004) Mol Cell 16,
269-279 41. Volkmann, N., Amann, K. J., Stoilova-McPhie, S., Egile, C., Winter, D. C., Hazelwood,
L., Heuser, J. E., Li, R., Pollard, T. D., and Hanein, D. (2001) Science 293, 2456-2459 42. Pollard, T. D., Blanchoin, L., and Mullins, R. D. (2000) Annu Rev Biophys Biomol Struct
29, 545-576 43. Li, R. (1997) J Cell Biol 136, 649-658
10
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure Legends
Figure 1 Yeast actin polymerization, but not muscle actin polymerization, is enhanced by yeast Arp2/3 in the absence of an Arp2/3 activating factor Yeast ( , ) and muscle G-actin (◊, ) at 1 µM concentration in the absence ( , ◊) and the presence ( , ) of 50 nM Arp2/3 was induced to polymerize by the addition of salt as described in Experimental Procedures. Polymerization was monitored by the change in light scattering (L.S.) over time with excitation and emission wavelengths at 365 nm as described in Experimental Procedures. The experiment was repeated in triplicate with the same results. Figure 2 Acceleration of muscle actin polymerization by yeast Arp2/3 complex requires the Las17p WA. Muscle actin, 2 µM ( ), with 50 nM yArp2/3 in the presence of Las17p WA at either 50 nM ( ), 100 nM ( ), or 200 nM (◊), is induced to polymerize by the addition of salt. Polymerization is monitored by the increase in light scattering as a function of time as in Fig. 1. The same result was obtained in duplicate experiments. Figure 3 The degree of acceleration of yeast actin polymerization depends on yArp2/3 concentration and the presence of Las17p WA. Panel A, 1.2 µM of 10% pyrene-labeled yeast actin is polymerized in the presence of yArp2/3 at 0 nM ( ), 25 nM ( ), 50 nM ( ), and 100 nM (◊). Panel B, 1.2 µM yeast actin is polymerized alone ( ), in the presence of either 100 nM Las17p WA ( ), 25 nM yArp2/3 ( ), or 100 nM Las17p WA and 25 nM yArp2/3 (◊). In both panels, polymerization is induced by as in Fig. 1 and is followed by the increase in pyrene fluorescence over time as described in Experimental Procedures. The experiment was repeated in triplicate with the same results. Figure 4 Different morphologies of branched actin filament structures produced by yeast Arp2/3 complex. 2 µM actin was polymerized in the presence of 25 nM Arp2/3 with or without 500 nM Las17p WA. Aliquots of the samples were removed at different time points, filaments was stabilized by adding rodamine phalloidin, and then the samples were monitored with a fluorescence microscope as described in Experimental procedures. The scale bar is 2 µm. . Panel a, c, g, and i; muscle actin. Panel b, d, e, f, and h; yeast actin. Figure 5 Persistence of yeast Arp2/3-induced branched structures as a function of time in the presence and absence of Las17pWA fragment. Aliquots of polymerizing 2 µM actin solutions were removed at various times following induction of polymerized and visualized as described in Experimental Procedures. For quantitation, a filament was counted as a single branched filament regardless of the number of branches attached to it. For each sample, the percent of branched filaments, independent of morphology, was calculated based on over 700 filaments per sample, and the values were plotted as shown in panels A (yeast actin with yArp2/3 alone), B (yeast actin with yArp2/3 and Lasp17p WA), and C (muscle actin with yArp2/3 and Lasp17p WA). Error bars in panels A and C, showing the mean and standard deviations, are based on three independent experiments. The curve in panel B is based on the average of results obtained in two independent experiments. Figure 6 The population analysis of branches originating from newly polymerized or pre-formed filaments. C374-Alexa-488 labeled 2 µM preformed yeast F-actin, stabilized by
11
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
phalloidin, was mixed with 2 µM G actin in the presence of 25 nM yArp2/3 and 500 nM Las17p WA in G-buffer containing 2 mM MgCl2 and 50 mM KCl. Rhodamine phalloidin was added to the reaction solution after 3 min, and a diluted sample was examined by fluorescence microscopy. The schemes drawn in the X-axis depict the categories of branched filament formations observed. The open bar represents a preformed filament and a solid bar denotes a newly formed filament, either an extension from an old filament or a branch. Figure 7 The kinetics of Pi release from polymerizing yeast actin in the presence of yArp2/3 with or without Las17p WA. The polymerization of yeast actin (1.2 µM) alone (─ ─), in the presence of 25 nM yArp2/3 (─ ─), or in the presence of 25 nM yArp2/3 and 500 nM Las17p WA (─ ─) was monitored by light scattering. In parallel reactions, the kinetics of Pi release was monitored with the EnzChekTM assay depicted by the same symbols but without interconnecting lines. The same result was obtained in duplicate experiments. Figure 8 Polymerization of V159N and WT actins in the presence of yArp2/3. Yeast V159N actin ( , ◊) and WT actin ( , ) both at 1.2 µM either alone ( , ) or in the presence of 25 nM yArp2/3 ( , ◊) were induced to polymerize by the addition of salt. The polymerization was monitored by the change of light scattering signal. The same result was obtained in duplicate experiments. Figure 9 Persistence of yeast Arp2/3-induced branched V159N actin structures as a function of time. The experiment described in Fig. 5 was repeated with 2 µM V159N actin and 25 nM yArp2/3 in the absence of Las 17p WA. Figure 10 Polymerization of yeast WT and V159N actins in the presence of bArp2/3 and N-WASP. Panel A, yeast WT actin,t 2.5 µM, was polymerizes alone ( ), in the presence of either 25 nM yArp2/3 ( ), 25 nM bArp2/3 ( ), or 25 nM bArp2/3 and 500 nM N-WASP (◊). Panel B, V159N actin polymerized alone ( ), in the presence of either 25 nM yArp2/3 ( ), 25 nM b Arp2/3 ( ), or 25 nM b Arp2/3 and 500 nM N-WASP (◊). The polymerization was observed by the increase of light scattering signal. The same result was obtained in duplicate experiments.
12
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Kuo-Kuang Wen and Peter A. Rubensteinan ARP2/3 activating protein
Acceleration of yeast actin polymerization by yeast ARP2/3 complex does not require
published online April 27, 2005J. Biol. Chem.
10.1074/jbc.M502024200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on March 4, 2020
http://ww
w.jbc.org/
Dow
nloaded from
![[6] Prota Inggris](https://img.pdfslide.net/doc/110x75/55cf97c2550346d033936e17/6-prota-inggris-5621429114806.jpg)
![[6] PROTA PAI](https://img.pdfslide.net/doc/110x75/5572137f497959fc0b926b70/6-prota-pai-55c99c6fe75b6.jpg)
![[6] PROTA PKN](https://img.pdfslide.net/doc/110x75/55cf8a8755034654898b7086/6-prota-pkn-56d4e8d1a871c.jpg)
![[6] PROTA TIK](https://img.pdfslide.net/doc/110x75/55cf880455034664618c7818/6-prota-tik.jpg)
![[6] PROTA SBK](https://img.pdfslide.net/doc/110x75/563dba10550346aa9aa26259/6-prota-sbk.jpg)
