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Address correspondence to Gregory S. Payne, Dept. of Biological Chemistry, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095-1737. Tel.: (310) 206-3121. Fax: (310) 206-5272. E-mail: gpayne{at}mednet.ucla.edu
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Abstract |
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Key Words: endocytosis; SLA1; NPF; adaptor; clathrin
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Introduction |
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Common examples of mammalian endocytic targeting signals include tyrosine-based motifs, YXX and NPXY (where X is any amino acid [aa]* and
is a bulky hydrophobic aa), and di-leucine motifs, all of which act as signals for packaging into endocytic clathrin-coated vesicles (Kirchhausen et al., 1997; Marks et al., 1997). In the best-characterized case, YXX
motifs interact with the µ subunit of the heterotetrameric AP-2 adaptor complex (Ohno et al., 1995). AP-2 also binds to clathrin, promoting assembly of individual clathrin molecules into a polyhedral scaffold (Kirchhausen, 1999). By serving as an adaptor linking receptors to the clathrin coat, AP-2 is thought to couple cargo recruitment with vesicle formation. A variety of other endocytic targeting sequences likely functions in a similar fashion by direct or indirect interaction with AP-2 (Kirchhausen et al., 1997; Kirchhausen, 1999). In a possible exception, the NPXY motif has been reported to interact directly with clathrin (Kibbey et al., 1998).
In the yeast Saccharomyces cerevisiae, two distinct classes of clathrin-facilitated endocytic targeting signals have been uncovered, one containing critical lysine residues, and the other consisting of the sequence NPFX(1,2) (NPFX[1,2]D) (Rohrer et al., 1993; Tan et al., 1996). The lysine-based signal relies on ubiquitylation subsequent to local serine and threonine phosphorylation (Hicke and Riezman, 1996; Hicke et al., 1998). Mono-ubiquitylation is sufficient to trigger endocytosis, in contrast to the polyubiquitin chains of four or more residues required for proteasome-mediated degradation (Chau et al., 1989; Galan and Haguenauer-Tsapis, 1997; Roth et al., 1998; Terrell et al., 1998; Thrower et al., 2000). Expanding evidence suggests that ubiquitin acts as an endocytic targeting signal in mammals as well as yeast, although the mechanism by which this signal directs cargo to the endocytic machinery is currently unclear (Hicke, 2001).
The NPFX(1,2)D signal was originally discovered in the cytoplasmic domain of the furin-like protease Kex2p (Tan et al., 1996). Kex2p cycles between the Golgi complex and endosomes without normally encountering the cell surface (Wilcox et al., 1992; Brickner and Fuller, 1997). Despite this itinerary, the Kex2p cytoplasmic domain directs rapid endocytosis when fused to an endocytically inactive version of a cell surface protein, the -factor mating pheromone receptor, Ste2p (Tan et al., 1996). Mutational analysis of the Ste2p/Kex2p chimeric protein defined the sequence NPFSD as the endocytic targeting signal. Characterization of a similar sequence, NPFSTD, naturally present in Ste3p (the cell surface receptor for
-factor mating pheromone), established NPFX(1,2)D as a motif that can direct rapid internalization of an integral membrane protein that normally transits the plasma membrane.
The NPFX(1,2)D motif shares sequence features with the mammalian NPXY endocytic targeting signal. Furthering this resemblance, phenylalanine can function in place of tyrosine in the NPXY signal (Davis et al., 1987). In addition, both motifs can direct clathrin-mediated uptake, raising the possibility that the two signals might function by similar mechanisms (Davis et al., 1986; Tan et al., 1996). An alternative model derives from identification of NPF as a motif recognized by proteins bearing Eps15 homology (EH) domains (Salcini et al., 1997). Involvement in endocytosis is a common characteristic of EH domain proteins, including Eps15 and the yeast proteins Pan1p, End3p, and Ede1p (Benedetti et al., 1994; Wendland et al., 1996; Carbone et al., 1997; Benmerah et al., 1998; Gagny et al., 2000). Accordingly, EH domain proteins are candidate endocytic adaptors for the NPFX(1,2)D signal.
Here we present in-depth mutational analysis of the NPFX(1,2)D signal, indicating a closer relationship with EH domain ligands than with the NPXY endocytic targeting signal. However, the NPFX(1,2)D motif is not efficiently recognized by yeast EH domain proteins. Instead, we identified the actin-associated protein Sla1p as the component of the endocytic machinery that recognizes NPFX(1,2)D. Sla1p binds NPFX(1,2)D through a domain unrelated in sequence to EH domains. Our results suggest that Sla1p represents a novel type of endocytic adaptor that can link NPFX(1,2)D-containing cargo to the actin and clathrin-based endocytic machinery in yeast.
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Results |
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In the context of the original Ste2p/Kex2p chimera, the aspartate in NPFSD is an important component of the signal, although an aspartate to alanine mutant displayed a residual level of endocytosis (Tan et al., 1996). We tested the role of aspartate by substituting either alanine or glutamate in Ste2p318-NPFSD. In this receptor, the alanine mutation reduced endocytosis to background levels (Fig. 1 C). More importantly, the glutamate mutation was also disruptive (Fig. 1 C), establishing that aspartate, not simply a negatively charged side chain, is required for full endocytic targeting activity.
Serine phosphorylation is required for ubiquitylation of the yeast lysine-based endocytic targeting signal and several serine and/or threonine kinases have been implicated in endocytosis (Hicke et al., 1998; Cope et al., 1999; Zeng and Cai, 1999). Serine and/or threonine residues are located within and surrounding the NPFX(1,2)D signals derived from Kex2p and Ste3p. To determine whether phosphorylation might influence efficacy of the NPFX(1,2)D motif, serine and threonine residues were converted to alanine in Ste2p318-NPFSD. In the same construct, the glutamate preceding the NPFSD motif was also replaced with alanine. Alternatively, serine and threonine residues were changed to aspartate to mimic phosphoserine. Endocytosis of both mutants was normal (unpublished data), arguing that local phosphorylation/negative charge is not important for NPFX(1,2)D targeting activity.
Native Ste2p contains a functional NPFX(1,2)D-like motif
Ste2p contains a sequence related to the NPFX(1,2)D signal, GPFAD, beginning at residue 392 in the COOH-terminal cytoplasmic domain. We examined the endocytic targeting potential of this sequence in the context of the native receptor by introducing alanine (inactivating) or asparagine (possibly activating) mutations at G392. The alanine mutation did not affect Ste2p endocytosis (Fig. 2 A). However, G392N resulted in a reproducible enhancement in uptake kinetics at the 5-min time point (Fig. 2 A), suggesting that the sequence contributes to endocytosis.
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Although Ste2p7KR does not appear to be ubiquitylated, it was formally possible that the remaining cytosolic lysines could act as low-level or transient ubiquitin acceptors. To assess this possibility, the G392A and G392N mutations were analyzed in a form of Ste2p in which arginines replaced all 11 cytoplasmic lysines (Ste2p11KR).
Ste2p11KR and Ste2p11KR-G392N were internalized 1.5-fold more efficiently than the cognate 7KR versions (Fig. 2 C; unpublished data), for reasons that are currently unclear. However, internalization remained sensitive to the G392A mutation (Fig. 2 C). Thus, NPFAD and GPFAD function in the absence of cytoplasmic lysines, indicating that NPFX(1,2)D-mediated endocytosis does not require cargo ubiquitylation.
Collectively, these data provide evidence that an NPFX(1,2)D-related motif participates redundantly with ubiquitin in directing endocytosis of Ste2p. Although GPFAD directs endocytosis, glycine is less optimal than asparagine at the first position. Consequently, Ste2p11KR-G392N and Ste2p-G392A represent sensitive reporters to distinguish NPFX(1,2)D- and ubiquitin-dependent modes of endocytosis. Henceforth, we will refer to these reporters as Ste2p-NPFXD and Ste2p-Ub, respectively.
NPFSD interacts with Sla1p
To isolate an endocytic adaptor for the NPFX(1,2)D signal, we carried out a yeast two-hybrid screen. We generated a bait that carried the cytoplasmic domain sequences from Ste2p318 fused to three tandem copies of the 11-residue Kex2p-derived NPFSD sequence (Fig. 3 A). To eliminate activation of reporter genes by the NPFSD bait alone, glutamates in the Kex2p derived sequences were altered to alanine, a mutation that does not affect NPFX(1,2)D-mediated endocytosis (Tan et al., 1996). We reasoned that the triple repeat might improve chances of detecting interactions, as the initial internalization rate of Ste2p
318-NPFSD was enhanced
2.5-fold by adding two additional copies of the Kex2p NPFSD-containing sequence (unpublished data). Using the triple-repeat bait we identified a plasmid carrying a fragment of the SLA1 gene, but no plasmids expressing EH domain proteins. SLA1 encodes a 1,244-aa protein implicated in actin dynamics (Holtzman et al., 1993; Ayscough et al., 1997, 1999). Furthermore, Sla1p interacts with Pan1p and End3p, two proteins that function in actin organization and endocytosis, suggesting a role for Sla1p in the endocytic process (Tang et al., 2000). Sla1p contains three NH2-terminal SH3 domains, a central region with a proline rich sequence flanked by two domains unrelated to each other but conserved in other fungal and plant species (SLA1 homology domains [SHDs]), and multiple repeats of the sequence TGGXXXPQ in the COOH-terminal domain that potentially serve as phosphorylation sites (Fig. 3 B) (Ayscough et al., 1999; Zeng and Cai, 1999). The fragment isolated in the two-hybrid screen spans aa 4711185, lacking the SH3 domains.
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We also tested NPFSD for interaction with each of the five EH domain-containing proteins encoded in S. cerevisiae. These proteins were of particular interest because of sequence similarities between the NPFX(1,2)D signal and EH domain ligands, and because three of these proteins (Pan1p, End3p, and Ede1p), are involved in endocytosis (Benedetti et al., 1994; Wendland et al., 1996; Gagny et al., 2000). Using the two-hybrid assay, only the SLA1 fragment displayed interaction with NPFSD (Fig. 3 C). In contrast, Pan1p interacted with Yap1801p and Yap1802p, two proteins with five NPF motifs each, none containing an aspartate at position +5 and only one containing an aspartate at +6 (Fig. 3 D) (Wendland and Emr, 1998). Ede1p and End3p interacted with Yap1802p but not Yap1801p (Fig. 3 D). These results imply that Sla1p uniquely recognizes the NPFX(1,2)D motif.
NPFX(1,2)D-mediated endocytosis requires SLA1
The role of Sla1p in NPFX(1,2)D-mediated endocytosis was investigated by evaluating the effects of disrupting SLA1 (sla1) on internalization of Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-Ub (Ste2p-G392A). Uptake of
-factor mediated by Ste2p-NPFXD was abolished in sla1
cells (Fig. 4 A). The effects of sla1
on Ste2p-G392N and Ste2p-Ub were far more subtle; after a 5-min lag, internalization of both receptors was essentially normal. Sla1p was also required for uptake directed by the native GPFAD sequence in Ste2p (Fig. 4 B). The selectivity of sla1
effects on Ste2p-NPFXD and Ste2p-GPFXD supports the hypothesis that Sla1p acts as a specific endocytic adaptor for the NPFX(1,2)D signal. However, the lag in ubiquitin-mediated endocytosis suggests Sla1p also participates more generally in the initiation of cargo internalization.
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Sla1p SHD1 is required for NPFX(1,2)D-mediated endocytosis
We designed a mutant of SLA1 lacking sequences encoding SHD1 (P492-G551, sla1-shd1) to examine the role of this domain in endocytosis. Centromere-based plasmids expressing either SLA1 or sla1-
shd1 were introduced into sla1
cells and Sla1p expression was monitored by immunoblotting. The results indicated that deletion of SHD1 does not destabilize Sla1p (unpublished data). We then compared endocytosis of Ste2p-G392N, Ste2p-NPFXD, and Ste2p-Ub in sla1-
shd1 cells to Ste2p-G392N endocytosis in wild-type cells. As shown in Fig. 6 A, internalization of Ste2p-G392N and Ste2p-Ub in sla1-
shd1 cells lagged only at the 5-min time point compared to the wild-type control. The delay was less pronounced than that observed for the same receptors in sla1
cells (compare Figs. 4 and 6 A). However, uptake of Ste2p-NPFXD was essentially eliminated (Fig. 6 A), as was uptake of Ste2p-GPFXD (unpublished data), equivalent to the effects of deleting the entire SLA1 gene (Fig. 4).
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Discussion |
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SHD1: a novel NPF-binding domain
Our results identify the Sla1p SHD1 as a novel NPF-binding domain, unrelated in primary structure to NPF-binding EH domains. Some basic characteristics of NPF recognition by SHD1 can be inferred from analysis of endocytic targeting by the NPFX(1,2)D signal and from two-hybrid interaction studies. Recognition can occur relatively independent of NPFX(1,2)D position within the cytoplasmic domain of the receptor constructs tested, as close as 20 aa from the membrane spanning domain in Ste2p318-NPFSD, or as far as 96 aa from the membrane spanning domain of Ste2p-NPFXD. Several sequence features of NPFX(1,2)D appear to be especially important for SHD1 binding. Previous alanine mutagenesis identified key residues at positions 13 and 5 in NPFSD (Tan et al., 1996). Our current results indicate a preference for asparagine over glycine at position 1, and strict requirements for phenylalanine rather than tryptophan and tyrosine at position 3, and aspartate instead of glutamate at position 5. The sequence requirements at positions 13 are similar to EH domain ligands and consistent with the type Ilike ß turn conformation observed in the solution structure of an NPF peptide bound to the second EH domain of Eps15 (de Beer et al., 2000). Accordingly, we suggest that the NPFX(1,2)D signal binds to SHD1 in a conformation similar to a type I ß turn with intimate hydrophilic contacts through the aspartate at position 5 or 6.
The NPFSD signal interacted with SHD1 but not EH domaincontaining proteins. For EH domains, residues flanking NPF can influence specificity for different domains (Paoluzi et al., 1998). Of the EH domains from yeast proteins implicated in endocytosis (Pan1p, End3p, and Ede1p), only the second EH domain of Pan1p preferentially selected NPFXD peptides from a phage display library (Paoluzi et al., 1998). However, this binding was reportedly weaker than other EH domainNPF interactions. Thus, aspartate may play a key role in determining the preference of NPFX(1,2)D for SHD1 rather than EH domains, although other surrounding residues may also contribute to binding specificity.
After discovery of the NPFSD endocytic targeting signal in Kex2p, we sought the motif in proteins that normally undergo endocytosis. Two such proteins, Ste2p and Ste3p, contain NPFX(1,2)D and ubiquitin-based signals, although the apparent contributions differ in the two receptors. In Ste2p, these two signals appear to function in both pheromone-stimulated and constitutive uptake (Hicke et al., 1998; unpublished data). In Ste3p, the NPFX(1,2)D signal is particularly important for pheromone-stimulated internalization (Tan et al., 1996). A search of the Saccharomyces Genome Database revealed seven established or putative plasma membrane proteins with the sequence [G,N]PFX(1-2)D, suggesting that the NPFX(1,2)D motif, either alone or with ubiquitin-based signals, potentially directs endocytosis of a subset of cell surface proteins. Diversification provided by NPFX(1,2)D signals could allow special regulatory inputs, or increase overall endocytic efficiency by contributing contacts to the endocytic machinery distinct from those of other signals. Furthermore, the targeting function of NPFX(1,2)D may not be limited to endocytosis, as the signal was first identified in Kex2p, a protein that cycles between the Golgi and endosomes without normally transiting the cell surface. Therefore, it is possible that Sla1p SHD1 interactions with NPFX(1,2)D signals play roles at multiple protein sorting steps.
Sla1p: an endocytic adaptor
SLA1 was originally uncovered in a screen for mutations that result in lethality when combined with a deletion of the gene encoding the actin binding protein Abp1p (Holtzman et al., 1993). Characterization of sla1 mutants and Sla1p interaction partners indicate a role for Sla1p in actin cytoskeleton dynamics (Ayscough et al., 1997, 1999; Li, 1997; Madania et al., 1999; Drees et al., 2001). Importantly, Tang et al. (2000) have provided evidence that Sla1p forms a complex with Pan1p and End3p, two proteins essential for endocytosis. Our results extend the connection of Sla1p to endocytosis by demonstrating the role of the SHD1 domain in NPFX(1,2)D-mediated internalization. Considering these findings, we propose that the Sla1p/Pan1p/End3p complex serves as an endocytic adaptor complex linking cargo to the actin and clathrin-based endocytic machinery in yeast (Fig. 7).
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Members of the Sla1p/Pan1p/End3p complex also interact with clathrin-associated proteins (Fig. 7). Through its second EH domain, Pan1p binds yeast epsins (Ent1p and Ent2p) and AP180s (Yap1801p and Yap1802p), presumably by recognizing NPF motifs within these proteins (Wendland and Emr, 1998; Wendland et al., 1999). Our two-hybrid results suggest that End3p can also associate with Ap1802p. In turn, yeast epsins and AP180s interact with clathrin, likely through COOH-terminal clathrin-binding motifs (Wendland and Emr, 1998; Wendland et al., 1999). Epsins and AP180s contain NH2-terminal ENTH domains, which in mammalian proteins have been reported to mediate binding to phosphatidyl 4,5 bisphosphate (Ford et al., 2001; Itoh et al., 2001; Mao et al., 2001). Considering these interactions in sum, the Sla1p/Pan1p/End3p complex can coordinate a network linking the vesicle coat protein clathrin to the plasma membrane and endocytic cargo. Mutations of clathrin generally have less severe effects on endocytosis than mutations affecting actin cytoskeleton components. However, clathrin inactivation decreases internalization rates of different receptors (including NPFX[1,2]D-dependent proteins) between two- and fivefold (Tan et al., 1993, 1996; Payne et al., 1988). These results, and the well-established role of clathrin in mammalian cell endocytosis, argue that clathrin coat formation at least facilitates endocytosis in yeast. Through the interactions shown in Fig. 7, the Sla1p/Pan1p/End3p complex has the potential to couple cargo collection to clathrin coat formation, similar to the mammalian AP-2 clathrin adaptor complex.
Database searches for homologues of Sla1p or the SHD1 domain identify significant matches in several fungal species and Arabidopsis, but not other organisms for which sequence information is available. However, there are provocative parallels between the Sla1p/Pan1p/End3p complex and a protein network coordinated by Eps15, raising the possibility of an analogous complex in mammals. Pan1p and Eps15 similarly consist of multiple NH2-terminal EH domains, central predicted coiled-coil regions, and proline-rich COOH termini, and both participate in endocytosis (Wendland et al., 1996; Carbone et al., 1997; Tang et al., 1997; Benmerah et al., 1998). Eps15 interacts with intersectin, a protein with two NH2-terminal EH domains, and five COOH-terminal SH3 domains that has also been implicated in endocytosis (Yamabhai et al., 1998; Sengar et al., 1999; Simpson et al., 1999). Like Sla1p, intersectin combines NPF recognition modules with SH3 domains. Furthermore, through its EH domains, intersectin recognizes NPFXD sequences in a cytoplasmic domain of the integral membrane protein, SCAMP1 (Fernandez-Chacon et al., 2000). Importantly, the NPFXD motifs in SCAMP1 can interact with intersectin EH domains, but not Eps15 EH domains, a selectivity mirroring preferential association of NPFX(1,2)D with SHD1 over Pan1p EH domains. Therefore, the intersectin EH domain and the Sla1p SHD1 domain may exemplify convergent evolution for ligand binding specificity, suggesting that Sla1p and intersectin may be, at least in part, functionally homologous.
As we have proposed for the Sla1p/Pan1p/End3p complex, the Eps15/intersectin complex has the potential to couple NPFXD-containing cargo to the actin cytoskeleton and clathrin-coats. One such cargo could be SCAMP1, as it cycles through the plasma membrane and interacts with intersectin through NPFXD sequences (Brand et al., 1991; Brand and Castle, 1993). Eps15 and intersectin each contain candidate acidic aa tryptophan ARP2/3 activation sequences. Additionally, a splice variant of intersectin contains a DBL homology domain that stimulates actin polymerization through Cdc42 nucleotide exchange activity (Hussain et al., 2001). Interactions with clathrin coats could be mediated by EH domains in both Eps15 and intersectin that bind epsins (Chen et al., 1998; Yamabhai et al., 1998; Sengar et al., 1999), and SH3 domains in intersectin that bind proline-rich domains of clathrin coat accessory factors (Roos and Kelly, 1998; Yamabhai et al., 1998; Sengar et al., 1999). Also, Eps15 binds AP-2 (Benmerah et al., 1995). Thus, the interaction networks radiating from the Sla1p/Pan1p/End3p and Eps15/intersectin complexes incorporate common functional targets. Based on the models derived from our studies, it should now be possible to test the extent of functional analogy between these components of the endocytic machinery in yeast and mammals.
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Materials and methods |
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Strains and plasmids
Table 1 describes the strains used in this study. Plasmids pEK3 (sst1::LYS2), a gift from Howard Riezman (University of Basel, Basel, Switzerland) and pAB506 (ste2::LEU2), a gift from James Konopka (University of Oregon, Eugene, OR) were used for single-step gene replacements (Rothstein, 1991) after cleavage with EcoRI and BamHI, respectively. SLA1 was disrupted by generating two overlapping PCR fragments and cotransformation. DNA was introduced into yeast strains by a lithium acetate protocol (Woods and Gietz, 2001).
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Yeast two-hybrid screen
Strain PJ69-4A was cotransformed with genomic DNA libraries in pGAD yeast two-hybrid vectors (James et al., 1996) and plasmid pGBD-NPFSDx3, and incubated on SD agar plates lacking adenine for up to 2 wk at 30°C. More than 2 x 106 transformants were plated for each reading frame, covering the entire library at a >99% confidence level. Bait plasmids from Ade+ His+ colonies were cured by growth in nonselective media, and prey plasmids isolated and sequenced by standard methods. Cells cured of bait vector were retransformed with either pGBD-NPFSDx3, or versions carrying mutations of the signal, and examined for growth on SD plates lacking adenine or histidine. No prey genes were isolated more than once, suggesting that the screen was not carried out to saturation. Other than SLA1, there were no other isolates likely to be involved in endocytosis or other protein trafficking steps.
Liquid ß-galactosidase assays were performed as described (Breeden and Nasmyth, 1987). Units were calculated as (1,000 x OD420)/([ml extract] x [incubation time {min}] x [protein concentration extract {mg/ml}]). To assess relative growth on agar plates, freshly transformed PJ69-4A cells were grown overnight to stationary phase in SDCAAtrp-leu media, diluted to 1 x 107 cells/ml, and then tenfold serially diluted. Dilutions were spotted onto SD plates with or without adenine and incubated at 30°C for 5 d. Images were collected on a SPEEDLIGHT PLATINUM gel documentation system (Lightools Research) and adjusted using standard settings in Adobe Photoshop (Adobe Systems).
Preparation of 35S-labeled -factor
Metabolically labeled -factor was prepared and purified from filtered culture supernatant over a cellulose-phosphate column essentially as described (Tan et al., 1993), using strain RK537-3B, a gift from Kendall Blumer (Washington University School of Medicine, St. Louis, MO).
35S-labeled -factor internalization assay
Metabolically labeled -factor was prepared as described in (Tan et al., 1993). The assay for binding and internalization of 35S-labelled
-factor was performed essentially as that described (Tan et al., 1996), except that internalization was carried out in 1% Bacto-yeast extract (Difco), 2% Bacto-peptone (Difco), 50 mM PO4, pH 6, 1% BSA, 20 µg/ml histidine, adenine and uracil) and dextrose was added to 5% to initiate endocytosis. Percent uptake is 100x the ratio of (internalCPM-backgroundCPM/boundCPM- backgroundCPM). All samples were measured in duplicate, and each experiment performed at least three times.
GST fusion affinity binding
pGEX-KG-NPFSDx3 and pGEX-KG-NPASDx3 were expressed in bacterial strain BL21 (DE3), and GST fusion proteins were affinity purified with glutathione-Sepharose (Amersham Pharmacia Biotech) as described in Smith and Johnson (1988). pET21b-SHD1 was induced by 0.1 M IPTG for 16 h at 18°C and cells were lysed by sonication. Lysates were incubated with GST-NPFSD or GST-NPASD beads overnight at 4°C. Bound proteins were resolved by SDS-PAGE on a 15% gel, and visualized by Coomassie blue stain or immunoblotting (Burnette, 1981) with anti-pentaHis antibody (QIAGEN) and secondary antibodies coupled to HRP (Bio-Rad Laboratories). HRP was visualized using enhanced chemilumenescence (Amersham Pharmacia Biotech). p926-SHD1 was expressed and purified as described (Pettit et al., 1998), except that protein expression was induced for 12 h at 18°C. To remove the NH2-terminal tag, purified protein was dialyzed into Tris buffer (20 mM Tris, 10% [vol/vol] glycerol, pH 8.0) using Spectra/Por dialysis membrane 2 (Spectrum), and then treated with rTEV protease (Invitrogen) for 1 h at 30°C in TEV cleavage buffer (50 mM Tris, 0.5 mM EDTA, and 1 mM DTT. pH 8.0). KCl was added to a final concentration of 100 mM, and then cleaved tag and recombinant protease were removed by incubation with Ni-NTA beads (QIAGEN) for 20 min at 4°C. Binding experiments with GST fusion proteins were carried out as above, except that the incubation step was performed for 20 min at 4°C. Samples were resolved by SDS-PAGE analysis on a 20% gel (Giulian et al., 1983) and visualized by Coomassie blue staining. Images were collected by laser densitometry and adjusted using standard settings in Adobe Photoshop.
Temperature-sensitive growth assay
Cells grown to stationary phase in SDCAA media at 30°C were diluted to 1 x 106 cells/ml, and then tenfold serially diluted. Dilutions were spotted onto SD plates and incubated at 30°C or 37°C for 3 d.
Phalloidin staining of yeast cells
Phalloidin staining of actin was carried out as described (Pringle et al., 1989). Staining was visualized using a 100x oil immersion lens on a Nikon FXA fluorescence microscope. Images were collected using a Photometrics cooled charge-coupled device camera and Isee software from Inovision and adjusted using standard settings in Adobe Photoshop.
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Footnotes |
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* Abbreviations used in this paper: aa, amino acid; EH, Eps15 homology; GST, glutathione-S-transferase; SHD, SLA1 homology domain.
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Acknowledgments |
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J. Howard was supported by the Jonsson Cancer Center Predoctoral Fellowship, UCLA Dissertation Year Fellowship, and is a fellow of the UCLA Medical Scientist Training Program. This work was supported by National Institutes of Health grant GM 39040 to G. Payne.
Submitted: 4 October 2001
Revised: 6 March 2002
Accepted: 12 March 2002
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