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Vol. 18, Issue 8, 2893-2903, August 2007
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*Department of Biology, The Johns Hopkins University, Baltimore, MD 21218; and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Submitted May 11, 2007;
Accepted May 11, 2007
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2005; Merrifield et al., 2004). These spatiotemporal colocalization studies correlate the function of a protein with the timing of its appearance in the process. In Saccharomyces cerevisiae, these endocytic events occur at dynamic cortical patches that contain actin and other proteins (Engqvist-Goldstein and Drubin, 2003); the proteins seen early at patches (Las17, Sla1, Sla2, and Pan1) bind to cargo, membrane phospholipids, and clathrin and function in the initial cargo selection and vesicle formation steps. The later arriving proteins (Myo5, Abp1, and Arp2/3 complex) interact with early proteins and function in the later steps of endocytosis, such as actin dynamics and vesicle scission (Kaksonen et al., 2003; Jonsdottir and Li, 2004).
Pan1 is an endocytic scaffold protein that is found at early patches, remains throughout patch maturation, and moves inward from the cell cortex as the new vesicle is formed and released (Kaksonen et al., 2003). PAN1 is essential in yeast, and Pan1 interacts with many different endocytic proteins (Figure 1A) (Sachs and Deardorff, 1992; Wendland et al., 1996). The functions of its binding partners suggest that Pan1 is a central factor in regulating transitions from early-to-late events in endocytic internalization. For example, the amino (N)-terminal region of Pan1 binds to several different proteins that are known or suspected cargo adaptor proteins: Sla1 (Tang et al., 2000), the yeast epsins Ent1 and Ent2 (Wendland and Emr, 1998), and the yeast CALM homologues Yap1801 and Yap1802 (Wendland et al., 1999), which are all predicted to act in early stages of internalization. The carboxy (C)-terminal region of Pan1 contains a region that binds to filamentous actin and an acidic (A) domain that binds to the Arp2/3 complex. Together, these domains cooperate to stimulate Arp2/3 complex activity and to promote actin polymerization (Duncan et al., 2001; Toshima et al., 2005). The later events in internalization, possibly including vesicle scission, involve actin polymerization (Yarar et al., 2005) and Arp2/3 complex recruitment (Merrifield et al., 2005). The central region of Pan1 mediates the formation of Pan1–Pan1 complexes (Miliaras et al., 2004), and also seems to link the N-terminal and C-terminal functions of Pan1 into a single protein complex (Miliaras et al., 2004; Toshima et al., 2005). Thus, Pan1 is poised to coordinate and regulate the sequence of early-to-late events necessary for efficient endocytic internalization.
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Type I myosins are highly conserved actin-based motors that participate in polarized morphogenesis, cell migration, and endocytosis (Geli and Riezman, 1996; Brown, 1997). Typically, myosins have N-terminal motor domains that bind actin in an ATP-dependent cycle and C-terminal tails that bind cargo through protein interaction domains. Type I myosins have an SH3 domain in their C-terminal tails, and fungal type I myosins also have an A domain at the extreme C-terminus that binds and activates the Arp2/3 complex (Geli and Riezman, 1996; Goodson et al., 1996; Evangelista et al., 2000; Lechler et al., 2000). MYO3 and MYO5 are the only genes encoding type I myosins in the Saccharomyces cerevisiae genome. Deletion of either gene does not affect growth; however, a double mutant is severely compromised (Goodson et al., 1996; Lechler et al., 2000). myo5 cells, but not myo3 cells, have a temperature-sensitive endocytosis defect, indicating that Myo5 may be more competent in endocytosis than Myo3 (Geli and Riezman, 1996). Myo3 and Myo5 both localize to cortical actin patches (Goodson et al., 1996), and a recent study reports that Myo5 appears transiently at cortical endocytic patches, immediately preceding the fast movement of vesicles away from the membrane, suggesting a function in vesicle scission (Jonsdottir and Li, 2004). Both the motor and Arp2/3 activities of Myo5 have been implicated in completing the late stages of endocytosis (Sun et al., 2006). In support of their endocytic roles, the C-terminal tails of Myo3 and Myo5 interact with multiple endocytic proteins: Las17, a yeast homologue of Wiskcott Aldrich Syndrome protein (WASP) (Evangelista et al., 2000); Vrp1, a yeast homologue of human WASP-interacting protein (Anderson et al., 1998); and Arc19 and Arc40, components of the Arp2/3 actin nucleating complex (Evangelista et al., 2000; Lechler et al., 2000). Vrp1 is an activator of the Arp2/3 actin polymerization activity of type I myosins in both S. cerevisiae and Schizosaccharomyces pombe (Sirotkin et al., 2005; Sun et al., 2006).
As more proteins are temporally placed along the timeline of endocytic events, a mechanistic understanding of the functions of each protein is needed to develop a complete model of endocytosis. This study describes our investigation into the function of the Pan1 PRD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Yeast Strains and Plasmids
Standard methods were used for growth, sporulation, tetrad dissections, DNA manipulations, and transformations of yeast (Burke and Stearns, 2000). The yeast strains used in this study are listed in Table 1. BWY2043 (Pan1-GFP), BWY2319 (Myo3-RFP), and BWY2405 (Myo5-RFP) strains were generated by C-terminal chromosomal integration of polymerase chain reaction (PCR) products as described in Longtine et al. (1998). The monomeric red fluorescent protein (RFP) plasmid used for the RFP tagging was a gift from W. Huh (UCSF) (Huh et al., 2003). BWY2510, contains pan1PRD-NAT in a synthetic genetic array (SGA) competent strain, was constructed by PCR-based double integration of a premature stop codon, with endogenous 3' untranslated sequence by using (5'-gaagaagcaaagattggtcatcctgatcatgcacgtgct TGA agaatttaatttgctttctaagatacg-3') and (5'-GTGACCCGGCGGGGACGAGGCAAGCTAAACAGATCTtgaagctgtaaaggaagaattggatcg-3') along with a NAT resistance (Goldstein and McCusker, 1999) cassette by using (5'-AGATCTGTTTAGCTTGCCTCGTCC-3') and (5'-cagaaattagtatacatacgtatctatagaaagcaaattaaatctGAATTCGAGCTCGTTTTCGACACTG-3'). The plasmids used in this study are listed in Table 2. DNA manipulations were performed using standard techniques. All restriction enzymes were purchased from New England Biolabs (Beverly, MA).
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). Sections were cut on a Reichert Ultracut T ultramicrotome, poststained with uranyl acetate and lead citrate, and observed on a Philips TEM 420 at 80 kV. Images were captured with an Olympus Soft Imaging Solutions (Münster, Germany) Megaview III digital camera.
Genetic Analysis
SGA analysis was performed as described previously (Tong and Boone, 2006). When using the temperature sensitive allele library, the cells were grown at room temperature (22°C) and for 1 d longer at each step. For the final double mutant selection, three sets of plates were pinned and grown at 22, 26, and 30°C.
Yeast Two-Hybrid Screening and Quantitative 
-Galactosidase Analysis
A two-hybrid screen (Phizicky and Fields, 1995) was performed by transforming the FRYL library (Fromont-Racine et al., 1997) into AH109 cells (Clontech, Mountain View, CA) containing pBW530, a plasmid of Gal4-DNA-binding domain fusion of Pan1p-APRD (amino acid [aa] 1232–1480), and grown on YNB-TRP-LEU-HIS-ADE media. From 
750,000 transformants, 25 positive clones were obtained, and the activation domain library plasmid was rescued and confirmed by retransforming into AH109 with pBW530 before being sequenced. For quantitative analysis, SFY526 cells transformed with a Gal-DNA–binding domain plasmid (pBW530) and transcriptional-activation domain plasmid were grown in selective liquid culture to mid-log phase and assayed for the expression of Galp-LacZ as described previously (Jarvis et al., 1988). Each value represents the average and SD for three independent quantifications.
Recombinant Pull-Down Experiments
Bacterial lysates were prepared from Rosetta cells containing either a pGEX-based glutathione S-transferase (GST)-fusion plasmid or a pET28c-based His6-fusion plasmid. Cells were grown to 0.6 ODs in SB media containing 34 µg/ml chloramphenicol and 50 µg/ml carbenicillin or 30 µg/ml kanamycin, respectively, and then they were induced with 0.1 mM isopropyl -D-thiogalactoside for 3 h at 37°C. Cells were harvested by centrifugation, washed, and resuspended in lysis buffer [20 mM Tris, pH 7.5, 1 mM EDTA, and 4-(2-aminoethyl)benzenesulfonyl fluoride protease inhibitors]. Lysis was achieved by a slow freeze at –20°C and a gentle thaw to room temperature. Then, lysozyme was added to 400 µg/ml and the lysates were incubated and mixed on a Nutator at room temperature for 20 min, finished with a final step of sonication on ice. The lysates were cleared with a 15,000 rpm in an SS-34 rotor (Sorvall, Newton, CT) at 4°C, and KCl was added to 150 nM and 0.2% Tween 20. Lysates were stored at –80°C. Thawed His6 or GST construct lysates were incubated with nickel agarose beads (QIAGEN, Valencia, CA) or glutathione agarose beads, respectively, for 1 h at room temperature to allow for binding. Protein-bound beads were washed three times with phosphate-buffered saline (PBS), and then they were tested for approximately equal concentrations of protein-bound by SDS-polyacrylamide gel electrophoresis (PAGE), standard Coomassie staining, and immunoblot analysis. To test for binding between the PRD and SH3 domains, protein-bound beads were incubated with lysates of the target for 1 h at room temperature. Nickel beads with bound His6-PRD proteins were incubated with GST lysates and glutathione beast with GST-bound SH3 domains were incubated with His6-PRD lysates. Beads were subjected to gentle centrifugation and washed three times with PBS. The initial supernatants and bound protein pellets were denatured in Laemmli buffer and run on SDS-PAGE gels for immunoblotting.
Coimmunoprecipitation Experiments
DDY1810 cells transformed with a myosin-myc plasmid were grown to mid-log phase and dounced in lysis buffer (20 mM HEPES, pH 6.8, 0.2 M sorbitol, 50 mM KOAc, and 2 mM EDTA) with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Extracts were prepared from the P3 pellet to a concentration of 20 ODs/ml in lysis buffer with 1% Triton X-100. Immunoprecipitations were performed with 500 µl of extracts, containing 10 ODs, polyclonal antibody against Pan1 (gift from A. Sachs, UC Berkeley), and protein A-Sepharose beads (experiments were repeated 5 times for Myo5 and 2 times for Myo3). For immunoblot analysis, 0.5% of the extracts and 10% of the immunoprecipitations were loaded for detection by using a monoclonal mouse Myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Protein Purification
Rabbit skeletal muscle actin, hemagglutinin (HA)-tagged Arp2/3 complex, Myo5, and Vrp1 were purified as described previously (Martin et al., 2005; Toshima et al., 2005; Sun et al., 2006).
GST-Pan1 PRD was expressed in and purified from the protease-deficient DDY1810 yeast strain. Cells were washed, and cell pellets frozen in liquid N2 were resuspended in PBS-KCl buffer (20 mM phosphate, 150 mM NaCl, 100 mM KCl, and 2 mM dithiothreitol). The cells were lysed using an EmulsiFlex C-3 (Avestin, Ottawa, Ontario, Canada) for 5 min at pulses of 21,000 psi. The lysate was clarified at 30,000 x g for 10 min at 4°C. The lysate was incubated with glutathione agarose beads (Invitrogen) for 3 h at 4°C, washed with the PBS-KCl buffer, and the GST fusion protein was eluted with a 50 mM glutathione, 50 mM Tris, pH 7.4, solution. Protein concentrations were determined using SYPRO dye (Invitrogen) with bovine serum albumin as a standard. GST and GST-Pan1 PRD were dialyzed in 20 mM HEPES, pH 7.5, 1 mM EDTA, 50 mM KCl, and 5% (vol/vol) glycerol.
SDS-PAGE and Immunoblotting
Proteins were separated on polyacrylamide mini gels (7.5–15%) at 18–25 mA in SDS running buffer (3 mM SDS, 25 mM Tris base, and 192 mM glycine), and then they were transferred onto nitrocellulose membranes at 80 V for 90 min in cold transfer buffer (20% methanol, 0.0375% SDS, 48 mM Tris base, and 39 mM glycine). The membranes are blocked in 5% milk in Tris-buffered saline/Tween 20 (TBST) (10 mM Tris, pH 7.5, 0.25 M NaCl, and 0.025% Tween 20). Blots were incubated in the specified primary antibody, washed three times in TBST, incubated with secondary antibodies conjugated to horseradish peroxidase (Pierce Chemical, Rockford, IL), and diluted 1:5000 in milk solution for 45–60 min. Blots were washed again three times in TBST, and then they were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical) for 5 min at room temperature. The chemiluminescence was visualized on a Fluorochem 8000 chemiluminescence system (Alpha Innotech, San Leandro, CA).
Dual Fluorescence Microscopy
Fluorescent images were captured using a Sensicam QE charge-coupled device camera (Cooke, Romulus, MI) on an Axiovert 135 TV inverted microscope (Carl Zeiss, Jena, Germany) equipped with a 100x/1.4 numerical aperture objective, Ludl motorized filter wheels (Ludl Electronic Products, Hawthorne, NY), fluorescein isothiocyanate (FITC) and Texas Red filter sets (Semrock, Rochester, NY), and IPLab software (Scanalytics, Fairfax, VA). In the capture two-color movies, IPLab software was used to drive the Sensicam QE camera and motorized filter wheels (Ludl Electronic Products) with FITC and Texas Red filter sets paired with a 4,6-diamidino-2-phenylindole/FITC/Texas Red multiband dichroic (Semrock). For the live cell imaging, cells were grown to early log phase on rich medium plates containing excess adenine at 26 or 30°C. Cells were placed in 2 µl of complete minimal media on the surface of an uncoated glass coverslip, and then they were inverted onto a glass slide. All imaging was performed at room temperature. Image analysis was performed using National Institutes of Health ImageJ (http://rsb.info.nih.gov/ij/) or SlideBook software (Intelligent Imaging Innovations, Denver, CO).
Actin Nucleation Assays
Actin assembly was performed using 2 µM rabbit skeletal muscle actin (5% pyrene labeled) as described previously (Sun et al., 2006), and assembly was monitored using a FluoroMax 3 fluorometer (Jobin-Yvon Horiba, Edison, NJ) with an excitation and emission of 365 and 407 nm, respectively. Barbed end concentrations were calculated using the rate of actin polymerization at 50% polymerization and a rate constant of 8.7 µM–1 s–1, as described previously (Higgs and Pollard, 1999).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Strains carrying pan1-20 have been broadly used as a canonical endocytic mutant; however, the mechanistic nature of the mutation has not been characterized. To better understand this allele and to place the experimental findings of pan1-20 cells in a molecular context, we sequenced the PAN1 open reading frame from pan1-20 cells, and we identified a single nucleotide deletion of guanine 4285. This deletion causes a shift in the open reading frame of the protein starting at amino acid 1429, which occurs in the middle of the PRD. The mutant protein is eight amino acids longer than wild type; however, the last 60 residues differ from the wild-type protein (Figure 1B).
As reported previously, the pan1-20 allele causes lethality, endocytic defects, actin cytoskeletal abnormalities, and extended membrane invaginations at high temperatures (Wendland et al., 1996). However, the functional consequences of a significant frameshift mutation should be apparent at all temperatures, although higher temperatures may exacerbate certain effects or phenotypes. To more rigorously assess the status and stability of the pan1-20 mutant protein, we performed quantitative Western blotting of cell extracts prepared from wild-type and pan1-20 cells grown at either 26°C or shifted to 37°C for 3 h. This experiment showed that full-length, intact, pan1-20 protein was present at roughly 70% of wild-type levels at 26°C and at one third of wild-type levels at 37°C (Figure 2A). When we examined the phenotypes of pan1-20 cells at the permissive temperature of 26°C, we observed endocytic defects as seen by the stabilization of the endocytic cargo Ste6-GFP at the plasma membrane (Figure 2B; Liu et al., 2007) and mild actin phenotypes (Wendland et al., 1996). Morphological analysis by electron microscopy of pan1-20 cells grown at permissive temperature did not show extended invaginations. However, there were approximately twofold more membrane invaginations in pan1-20 cells (42 invaginations in 24 cells) than in wild-type cells (23 invaginations in 26 cells) (Figure 2C). These data show that the phenotypes of pan1-20 cells are indeed present at permissive temperatures, although in milder form than at the nonpermissive temperature, and that they are consistent with a defect in endocytic internalization at the point of vesicle scission. Thus, the C-terminus of Pan1 may have a distinct role in endocytosis.
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; Duncan et al., 2001; Miliaras et al., 2004). It is possible that the temperature-sensitive and endocytic defects of the pan1-20 mutation might be due to more than the loss of the C-terminal sequence within the PRD, perhaps through a subtle effect on protein stability or the additional nonnative C-terminal residues (Figure 2A). Nonetheless, our findings highlight a potential importance for the PRD. Many endocytic proteins are redundant or share overlapping functions with other proteins that can mask their individual contributions (Engqvist-Goldstein and Drubin, 2003). Therefore, to determine whether there is an in vivo role for the Pan1 PRD, we sought to identify genetic backgrounds that caused specific sensitivity to loss of the Pan1 PRD. A pan1PRD strain was engineered by introducing a stop codon at aa1310 and removing the coding sequence 3' of this site while maintaining 200 base pairs of the 3' untranslated region, followed by an NAT resistance marker. Western blots confirmed that the pan1PRD strain produced a truncated protein at levels similar to endogenous protein (Figure 3A).
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PRD strain against an unbiased library of temperature-sensitive KanMX-marked alleles. This collection was constructed by integrating previously characterized temperature-sensitive alleles into the S288C genetic background (Li and Boone, personal communication). The collection contains alleles of 348 genes that are either essential or severely sick as deletions, covering 25 of the 33 biological process designations curated in the gene ontology terms (see Supplemental Material). To identify synthetic lethal or synthetic sick double mutant genetic interactions, we performed an SGA analysis (Tong et al., 2001; Tong and Boone, 2006) against the library of temperature-sensitive alleles by using the pan1PRD query strain. Synthetic sick interactions were identified with las17-14 and act1-121, and they were subsequently confirmed by tetrad analysis (Figure 3B). Thus, this broad screen identified interactions with ACT1, which encodes actin, and with LAS17, the yeast WASP homologue, which controls the actin nucleating activity of the Arp2/3 complex and functions at endocytic sites. In cycloheximide chase experiments monitoring the internalization and degradation of endogenous Ste3 at the permissive temperature of 26°C, the double mutants had greater endocytic defects than the single mutants (Figure 3D).
We also used a focused set of haploid deletion strains, corresponding to 59 genes with known endocytic, actin, and membrane functions (see Supplemental Material). This second round of SGA analysis with pan1PRD revealed a synthetic lethal interaction with sla1 (Figure 3C). Sla1 binds to the C-terminus of End3 and to the N-terminus of Pan1; this complex performs unknown functions in endocytic internalization (Tang et al., 2000; Zeng et al., 2001). The interactions identified in these genetic studies highlight a role for the PRD of Pan1 in actin dynamics and endocytic internalization, which is consistent with the phenotypes observed in pan1-20 cells.
The Pan1 PRD Directly Interacts with the SH3 Domains of the Type I Myosins Myo3 and Myo5 In Vitro and In Vivo
To identify protein interaction partners for the PRD of Pan1, we performed a yeast two-hybrid screen with a fragment of Pan1 (aa1232–1480), containing both the A domain and the PRD. The A domain was included because constructs of the PRD alone proved to be self-activating in the two-hybrid system. From a genomic yeast two-hybrid library (Fromont-Racine et al., 1997), multiple clones of the type I myosins Myo3 and Myo5 were identified (Table 3). All of the clones obtained in the screen contained the C-terminal SH3 domain of the myosins (Figure 4A). To determine whether the interaction with Pan1 was through the SH3 domain, we made single point mutations at a conserved tryptophan residue in the myosin SH3 domain: W1157S in Myo3 and W1123S in Myo5. This mutation causes Myo3 mislocalization in vivo and reduces SH3 domain-dependent physical interactions of Myo3 with its other known partners in yeast (Evangelista et al., 2000; Geli et al., 2000). We have observed a similar mislocalization with the corresponding mutation in Myo5 (Barker and Wendland, unpublished data). The SH3 domain mutants did not interact with the C-terminus of Pan1 in the two hybrid assay (Table 4), demonstrating that the interaction with Pan1 is dependent on the SH3 domain of the type I myosins.
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A consensus sequence PxxxPPxxP has been reported as the ligand for the type I myosin SH3 domains, and a single predicted ligand is present within the last 12 residues of the PRD of Pan1 (Tong et al., 2002). To test whether this C-terminal ligand is important for the interaction between Pan1 and the myosins, we made a truncated construct, His6-Pan1 PRD12 (aa1310–1468), in which the last 12 amino acids were removed. The GST-SH3 domains did not bind this truncated His6-Pan1 PRD12 from bacterial lysates (Figure 4C). Consistent with this, the smaller proteolytic fragments of the affinity-purified N-terminally tagged His6-Pan1 PRD, which would be expected to lack the extreme C-terminal residues, also did not bind to the GST-SH3 domain proteins (Figure 4C). Together, these data demonstrate that the last 12 residues of Pan1 are required for the binding of Pan1 to the type I myosins.
The Pan1–myosin interaction was confirmed as a bona fide in vivo interaction through coimmunoprecipitation of both Myo3 and Myo5 with Pan1 (Figure 4D). Endogenous Pan1 was immunoprecipitated from cells containing either Myo3-myc or Myo5-myc encoded by a plasmid, and the bound myosins were detected using an anti-Myc antibody. This confirms that the interaction can occur in the context of the full-length proteins. Although the degree of coimmunoprecipitation was somewhat weak, these results are reproducible and consistent with a transient interaction in vivo (see below). Together, these data indicate that the Pan1 and the type I myosins interact directly in vivo.
Spatiotemporal Colocalization Analysis of Pan1 and the Type I Myosins
With the biochemical confirmation of an interaction between Pan1 and the type I myosins, we proceeded to examine the spatial and temporal nature of the interaction in living cells. Pan1 was tagged with GFP at its C-terminus by integration at the PAN1 locus. The dynamics of Pan1-GFP were consistent with previous reports, having a lifetime of 30–40 s and culminating with movement toward the interior of the cell, which is interpreted as invagination or vesicle scission (Kaksonen et al., 2003). Myo3 and Myo5 were tagged with monomeric RFP at their C-termini by integration at their respective chromosomal loci. The Myo5-RFP had a lifetime similar to that in previous reports (Jonsdottir and Li, 2004; Sun et al., 2006), and unlike Pan1-GFP, the Myo5-RFP remained at the cell cortex for its duration. We observed Myo3-RFP to behave similarly to Myo5-RFP, as expected, also showing no internal movement. Statistically, we found no difference between the behavior of the myosins; however, the Myo5-RFP signal had a greater intensity.
To characterize the spatial and temporal features of the Pan1–myosin interaction, we mated Pan1-GFP cells with cells expressing a myosin-RFP and observed the colocalization of green and red fluorescence in the resultant diploid cells. Experiments were performed with both Myo3-RFP and Myo5-RFP; however, we only show Myo5 because the behavior of the myosins was indistinguishable. In single, static images, Pan1-GFP and the myosin-RFPs have a partial colocalization (Figure 5A), with some patches showing colocalization and some patches containing only Pan1-GFP or Myo-RFP. However, when analyzed temporally by two-color time-lapse movies, complete colocalization was ultimately seen for all patches: each Pan1-GFP patch was joined by myosin-RFP in its lifetime (Figure 5, B and C) (n = 31 for Myo5-RFP; n = 9 for Myo3-RFP). These results are consistent with the relative timing of protein behavior reported by Sun et al. (2006). Interestingly, the myosin always colocalized with Pan1 just before its internal movement, and then the myosin stayed at the cell cortex, whereas Pan1 moved inward (Figure 5, B and C). The intensities of both Pan1 and the myosin dissipated around the same time (Figure 5C). The precise timing of colocalization behavior suggests that the Pan1–myosin complex is poised to play a role in vesicle invagination and/or scission.
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; Evangelista et al., 2000). To determine whether the PRD of Pan1 plays a role in the localization of the myosins, a Myo5-GFP plasmid was transformed into wild-type and pan1PRD cells. A fluorescent GFP signal was observed at the membrane in both cells, indicating that the PRD of Pan1 does not influence the recruitment of Myo5 to the plasma membrane. However, we did observe an alteration in the lifetime of Myo5-GFP at the cortex; 13 ± 1 s in wild-type cells versus 20 ± 2 s in pan1PRD cells (n = 20 spots for each cell type; paired Student's t test, p < 0.0001) (Figure 5, D and E). An increase in Myo5-GFP lifetime was also observed in pan1-20 cells at permissive temperature (data not shown). Having observed a physiological effect of the lack of the Pan1 PRD, we went on to measure the lifetime of Myo5-GFP in our other genetic backgrounds: act1-121, act1-121 pan1PRD, las17-14, and las17-14 pan1PRD. In the single act1-121 and las17-14 mutants, the lifetime of Myo5-GFP was longer than in wild-type cells; however, in double mutants combined with pan1PRD, the lifetimes were significantly increased (Figure 5E). This increase in the lifetime of Myo5 at the membrane suggests that some function of the type 1 myosin is perturbed or delayed as a result of the absence of the PRD of Pan1.
Pan1 PRD Enhances the Actin Activity of the Type I Myosins
The type I myosins are one of four Arp2/3-stimulating actin nucleation promoting factors (NPFs). In fluorescent actin polymerization assays, the myosins on their own are weak activators, and they require the presence of verprolin, Vrp1, for more potent activity (Sirotkin et al., 2005; Sun et al., 2006). Because Vrp1 interacts with the SH3 domains of the myosins, we wanted to test whether the SH3 domain interaction with Pan1 had any effect on the NPF activity of the myosins. However, Pan1 itself is an NPF, with its own A domain. Therefore, we purified GST-PRD lacking the A domain to assess the effect of the PRD of Pan1 in isolation. GST-Pan1 PRD affected neither Arp2/3 nucleation activity nor Myo5 weak NPF activity (Figure 6 and data not shown). The addition of Vrp1 increased Myo5 NPF activity approximately threefold. GST-Pan1 PRD further enhanced the Arp2/3 complex activation by the Myo5p–Vrp1p complex: 75 nM GST-Pan1 PRD consistently increased the Myo5-Vrp1 NPF activity by 1.4-fold and 100 nM increased the activity by 1.8-fold, over that of Myo5 with Vrp1 and of Myo5 with Vrp1 and GST. Thus, the enhancement of Myo-Vrp1 NPF activity increased with increasing amounts of GST-Pan1 PRD.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PRD. Together, these findings support a model in which the interaction of Pan1 PRD with the myosins contributes actin activity that is required for efficient endocytosis.
The most striking feature from the spatiotemporal studies of the Pan1–myosin interaction is the intriguing timing of the interaction: the myosin occurs late in the Pan1 patch lifetime, just before the inward motion of the Pan1 patch (Figure 5). The interaction is brief (6–8 s) and terminates with the inward movement of Pan1, whereas the myosin remains at the membrane cortex. The inward movement of Pan1 and other proteins is interpreted as corresponding to an invagination event followed by vesicle movement just after scission (Kaksonen et al., 2003; Kaksonen et al., 2005). The type I myosins remain at the cell surface during these events, and they are thought to be key factors in generating and moving actin filaments to produce invagination forces (Sun et al., 2006). Pan1 and the type I myosins are each actin NPFs for the Arp2/3 actin nucleation complex, although a recent study suggests that Pan1 has a low specific activity (Sun et al., 2006). Both of these actin NPFs also bind filamentous actin (Albanesi et al., 1985; Zot et al., 1992; Toshima et al., 2005), and together are predicted to act late during the endocytic process. Our data are consistent with a model in which Pan1 triggers positive feedback on the Myo5–Vrp1 complex to facilitate the formation of force-generating actin filaments that are properly organized and linked to the invaginating vesicle coat.
Pan1 is not the first, nor only, protein that interacts with the SH3 domains of the type I myosins. Many models of myosin's actin Arp2/3 activity depict the heterogeneity of ligand binding to the SH3 domain of the type I myosins as a means by which multiple copies of the Arp2/3 complex are assembled on the actin. The SH3 domain binding partner proteins typically have other interaction domains that could cluster additional actin activators; this may be important for the actin requirements in endocytosis. Pan1 is a good candidate for this role in vivo, because it binds to and activates the Arp2/3 complex in addition to binding Myo3/5. We do not presently know whether this could explain the mechanism by which the Pan1 PRD stimulates Myo5-Vrp1 activity, especially because in the in vitro actin polymerization assays only the PRD was present. It is possible that the GST–PRD proteins used for the in vitro studies dimerized via the GST moieties, thereby contributing to a local concentrating effect of actin binding and polymerizing functions.
Could there be a more specific role for a Pan1–myosin complex in invagination and scission? Actin NPF activity is unique to fungal type I myosins, as type I myosins from other species lack an A domain (Evangelista et al., 2000). In fungi, this actin activity of the myosins could supplant the requirement of a dynamin-like protein in neck elongation/vesicle scission, and simultaneously provide an actin component to the process as well. Consistent with this idea, it has been suggested recently that a role for dynamin in animal cells is to help create elongated tubules, whereas tension mediated by the actin cytoskeleton is required for the scission of these tubules (Itoh et al., 2005). Additionally, a recent report shows that Myosin IE interacts with dynamin and the 5' inositol phosphatase synaptojanin, further suggesting a physical and potential regulatory link between type I myosins and scission machinery (Krendel et al., 2007).
In mammalian cells, vesicle scission is known to involve the GTPase dynamin. However, yeast has no obvious dynamin-like protein implicated in endocytosis. Scission defects in yeast have thus far been monitored through morphological analysis of electron micrographs to observe accumulated plasma membrane invaginations. We found this phenotype in cells carrying the pan1-20 allele, which has a frameshift mutation within the PRD, and it should not interact with the type I myosins in vivo. A similar phenotype has been reported for a type I myosin allele, myo5E901A (Jonsdottir and Li, 2004), suggesting that both Pan1 and the type I myosins act at the same functional point in the endocytosis pathway.
Our SGA experiments provide further evidence consistent with an in vivo role for the Pan1 PRD in an actin-related late endocytic process. First, the two temperature-sensitive alleles we uncovered as having synthetic sick phenotypes, act1-121 and las17-14, are both intimately linked to actin polymerization functions. These are allele-specific interactions, because we recovered only one of 17 act1 alleles and one of three las17 alleles in the collection tested in our screen (Figure 3 and Supplemental Material). Phenotypes of the act1-121 allele are not well described in the literature (Wertman et al., 1992), but there are a couple of features of interest. First, mutations in this region of Act1 (E83A, K84A) generally have the most deleterious effects on endocytosis (Whitacre et al., 2001). Second, purified act1-121 protein does not efficiently polymerize into filaments in vitro (Miller and Reisler, 1995). Either hyperstable or slowly polymerizing F-actin filaments might be expected to alter the dynamics and regulation of the late stages of endocytosis. Third, act1-121 mutant diploid cells have the relatively unique phenotype compared with many other act1 alleles of hyperpolarized and elongated buds, a phenotype also associated with mutations in septins and the p21-activated kinase homologue Cla4 (Drubin et al., 1993; Richman et al., 1999; Versele and Thorner, 2004). Cla4 also regulates Myo3 and Myo5 activity (Wu et al., 1996). Therefore, through act1-121, our genetic findings for the pan1PRD tie into the genetics of the activity of type I myosins.
The las17-14 temperature-sensitive allele (W41E, L133S) also exhibited synthetic sickness when combined with pan1PRD. This LAS17 allele is in the collection of temperature-sensitive alleles used in the SGA analysis, but it has not been characterized in the literature. Comparisons of Las17 with WASP homologues suggest that one of the mutated residues, W41E, may correspond to the invariant tryptophan residue positioned from 23 to 25 residues away from the start of WH1/EVH1 domains; this is one of the aromatic residues implicated in the WH1/EVH1 domain binding to peptide ligands (Rong and Vihinen, 2000). WH1/EVH1 domains typically bind proline-rich peptides, and the N-terminal region of Las17 that contains the WH1 domain has been reported to bind to Vrp1/End5 (Naqvi et al., 1998). It will be interesting to know whether the las17-14 mutant protein is disrupted for Vrp1 binding or Arp2/3 activation; if so, this might explain the synthetic sickness we observe with our pan1PRD allele.
We also confirmed a synthetic lethality between sla1 and pan1PRD. Sla1 is a well-established Pan1 binding partner that is an endocytic adaptor and also is an inhibitor of Las17-dependent Arp2/3 stimulation (Tang et al., 2000; Howard et al., 2002; Rodal et al., 2003). It has been shown previously that the pan1-4 allele exhibits synthetic lethality when combined with sla1 (Tang et al., 2000); however, this allele corresponds to an extensive C-terminal truncation lacking hundreds of amino acids (Zeng et al., 2001), whereas our truncation is missing just the final 170 amino acids and preserves the F-actin and acidic Arp2/3 binding sites. Perhaps the combination of losing regulation of both Las17 and the type I myosins is particularly deleterious; for example, the Arp2/3 stimulatory activity of Las17 is required in cells lacking the Arp2/3 activity of the type I myosins (Evangelista et al., 2000; D'Agostino and Goode, 2005). Likewise, loss of Las17 regulation by deleting SLA1, along with altered Myo3 or Myo5 regulation by deleting the PRD of Pan1 could lead to irrevocably perturbed actin structures. Consistent with this idea, pan1-20 cells that also lack the key PRD region show exaggerated actin structures that seem to be either very thick cables or dramatically elongated cortical patches when grown at the nonpermissive temperature (Wendland et al., 1996). Alternatively, the pan1-20 and pan1PRD proteins may have other activities affected in addition to their altered myosin SH3 ligand, such as putative regulated interactions between the N-termini and C-termini of Pan1 (Miliaras et al., 2004; Toshima et al., 2005).
Interestingly, the original screen that identified the pan1-20 allele also identified a mutation in SHE4 (Wendland et al., 1996). She4 is a chaperone for type I and type V myosins and is essential for myosin function under stress conditions (Toi et al., 2003; Wesche et al., 2003). This screen did not aim to identify mutants affecting one specific stage of endocytosis; however, the coincidence of the same screen identifying both the pan1-20 allele and a factor important for the type I myosins could suggest that these two mutants in fact act at the same substage of endocytic internalization.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Banting and Best Department of Medical Research, Terence Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario M5S 3E1 Canada.
Address correspondence to: Beverly Wendland (bwendland{at}jhu.edu).
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