From the Institut für Genetik, Forschungszentrum Karlsruhe, D-76021 Karlsruhe, Germany
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ABSTRACT |
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Phox homology (PX) domains were originally identified as a common motif of about 120 amino acids found in the p40phox and p47phox subunits of the neutrophil NADPH oxidase (Phox) complex (8). This domain is conserved from yeast to human, and more than 400 examples have been annotated in sequence databases (e.g. SMART (9)). PX domain-containing proteins have been implicated in highly diverse functions such as cell signaling, vesicular trafficking, protein sorting, and lipid modification (reviewed in Refs. 10 and 11).
PX domains show relatively little sequence conservation, yet their structure appears to be highly conserved. All published crystal structures exhibit a clearly defined PI-binding pocket (1215). Although phosphatidylinositol-3-phosphate (PtdIns(3)P) is the primary target of PX domains, binding to phosphatidic acid, phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2), phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) has been reported as well (16, 17).
The yeast genome encodes 15 PX domain-containing proteins that are involved in vesicular trafficking, cell signaling, control of bud emergence and cell polarity, or are of largely unknown function (Table I). All yeast PX domains (except the sequence outlier Ypr097p) bind specifically to PtdIns(3)P with different affinities (18).
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A direct involvement of the PX domain in protein-protein interactions has been observed in the p47phox subunit of the neutrophil NADPH oxidase complex. p47phox is held in an inactive state by an intramolecular interaction between its C-terminal Src homology 3 (SH3) domain and its PX domain. Upon activation by invading microorganisms, p47phox becomes phosphorylated and the PX-SH3 interaction is disrupted (21, 22). The C-terminal SH3 domain interacts with a proline-rich region of the p47phox-PX domain (13).
Moreover, a recent large-scale two-hybrid screen has shown that one PX-only protein from yeast, Ypt35p (YHR105W), interacts with several proteins (23). In order to investigate a possible role of the PX domain as a bona fide protein interaction module, we used PX-domain only bait proteins (for all yeast PX domain-containing proteins) in a genome-wide two-hybrid screen to search for binding partners of this domain. Our results argue that the PX domain is a bona fide protein-protein interaction module and binds predominantly to membrane proteins that are involved in vesicular transport.
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EXPERIMENTAL PROCEDURES |
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Construction of the Gal4DBD-ORF fusions was performed by means of PCR and recombination (26) as described in Ref. 23. Transformation was performed using the lithium acetate procedure (27). Bait constructs were transformed into yeast strain PJ69-4 (23) and preys into PJ69-4a (25).
For strains expressing the Gal4DBD-ORF fusions, successful cloning was confirmed by DNA sequencing.
PX domains cloned into pGSTag (28) for expressing them as GST fusions were kindly provided by Jong W. Yu and Mark A. Lemmon (18).
The plasmid pGSTag-Ypt35Y123A was constructed using the Quick-ChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the following primers: CCA AAC CTA TAA ACG AGC CTC CGA TTT CGT CAG (forward), CTG ACG AAA TCG GAG GCT CGT TTA TAG GTT TGG (reverse), with mutated bases shown in bold.
Two-Hybrid Screens and Retests
An array containing most of the 6,000 Saccharomyces cerevisiae ORFs expressed as Gal4AD fusions was used to screen for interacting proteins (23). Haploid transformants expressing either a full-length Gal4DBD-ORF fusion protein or a Gal4DBD-PX-domain-only fusion protein were mated to the array. The resulting diploids were pinned with a Biomek 2000 Laboratory Automation Workstation (Beckman-Coulter, Fullerton, CA) onto selective media. Positive prey clones from a first-round screen were re-arrayed as quadruplicate copies and tested again for reproducibility. All retests were carried out at least eight times, and their results were used to assign "quality" scores to our positives: positives that were reproducible in at least eight out of 12 or more tests were assigned values of 3, positives that were found at least four times out of 12 (or more) tests were assigned scores of 2, and positives that were found two to three times were assigned values of 1. Some highly reproducible positives were classified as 1 if the results were difficult to interpret due to high background or other reasons. Two-hybrid positives were classified by Gene Ontology descriptors as provided by the Saccharomyces Genome Database (www.yeastgenome.org).
Protein Expression and GST-Pulldowns
Expression clones of pGSTag-GST-PX domain fusion proteins have been described previously (18, 28). The PX domains fused to GST corresponded to the following fragments: Bem1p- (amino acids 269418), Bem3p- (502637), Grd19p- (1162), Mdm1p- (776908), Mvp1p- (121258), Snx4p- (20170), Snx42p- (150316), Vps5p- (271397), Vps17p- (88234), and Ypt35- (65214). Proteins were expressed in Escherichia coli and purified on glutathione-Sepharose as recommended by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ).
Modified primers for Yip1p, Yif1p, and Yip4p, containing a T7 promoter and an eukaryotic translation inititation sequence, were used for in vitro transcription and translation using the TNTTM-coupled reticulocyte system in the presence of [35S]methionine (Promega, Madison, WI). PCR primers were as follows: Yip1p forward, GGA TCC TAA TAC GAC TCA CTA TAG GGA ACA GCC ACC ATG TCT TTC TAC AAT ACT AGT; Yip1p reverse, TCA GAC AAA AAT TAC CAT GAG; Yip1p-N terminus reverse, GAT CCC CGG GAA TTG CCA TGC CCA GCC AAA TCT GAA TCG TT; Yif1p forward, GGA TCC TAA TAC GAC TCA CTA TAG GGA ACA GCC ACC ATG TCT TAT AAT CCG TAC GCA; Yif1p-reverse, TCA ACC CAT TAA CCA CAT TAG; Yif1p-N terminus reverse, GAT CCC CGG GAA TTG CCA TGG TCC ATA ATT CGT TGC CAG TT; Yip4p forward, GGA TCC TAA TAC GAC TCA CTA TAG GGA ACA GCC ACC ATG TCA TAC GGA AGA GAA GAC; Yip4p reverse, TCA GAA CTT TCT GCC GTG GCT.
GST-PX domain fusion proteins or GST alone were coupled to glutathione-Sepharose beads (Amersham Pharmacia Biotech) and incubated with 5 µl of in vitro-translated proteins in pulldown buffer (20 mM Tris pH 7,6; 1 mM mercaptoethanol; 3 mM EDTA, 150 mM NaCl; 1% Nonidet P-40) for 2 h at 4 °C under rotation. Bound proteins were washed six times (for 10 min under rotation at 4 °C) with pulldown buffer at decreasing detergence concentration (1 x 1%, 4 x 0.1% and 1 x 0%), harvested in SDS-sample buffer, separated by SDS-PAGE, and analyzed by autoradiography.
Lipid Overlay Assay
Protein-lipid overlay assays were performed as previously described (29). Briefly, 1 µl of lipid solution containing 6.5 to 200 pmol of phospholipids dissolved in a mixture of chloroform/methanol/water (1:2:0.8) was spotted on to Hybond-C extra membrane and allowed to dry at room temperature for 1 h. The membrane was blocked in 3% (w/v) fatty acid-free BSA in TBST [50 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20] for 1 h. The membrane was then incubated overnight at 4 °C with gentle stirring in the same solution containing 5 µg of the indicated GST-fusion protein. The membrane was washed six times for 30 min in TBST, and then incubated for 1 h with 1:1,000 dilution of mouse anti-GST monoclonal antibody (Sigma-Aldrich, St. Louis, MO), washed again as before, then incubated for 1 h with 1:5,000 dilution of anti-mouse-horseradish peroxidase conjugate (Bio-Rad, Hercules, CA). Finally, the membrane was washed 12 times for 1 h in TBST, and membrane-bound GST-fusion protein was detected by ECL (Amersham).
Interaction Visualization Software
Interactions were visualized with Cytoscape (www.cytoscape.org) (30).
Detailed Protocols
More detailed protocols are available at itgmv1.fzk.de/www/itg/uetz/protocols/.
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RESULTS |
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Full-length Versus Domain Interactions
Of the 15 PX domain proteins in yeast, nine showed a total of 67 two-hybrid interactions when they were screened as full-length proteins (Table II). We did not find any reproducible interactions for five proteins (Bem3p, Mdm1p, Spo14p, Ykr078p, and Ypr097p). Snx41p was not investigated further because it turned out to be a very strong transcriptional activator, even under high 3AT concentrations.
When we used the isolated PX domains as bait, only those from Mvp1p, Snx42p, Vam7p, and Vps17p showed a total of 13 reproducible interactions (Table II).
Bem1p, Snx4p, and Vps5p interacted in our assays only as full-length proteins, that is, their PX domain did not interact with any other protein. This indicates that other domains of these baits mediate their interactions. Grd19p and Ypt35p were tested only as full-length proteins as they consist almost exclusively of their PX domains. However, their two-hybrid interactions were confirmed with PX-only constructs in vitro (see below). In one case, Mvp1p, we did not find any interactions for the full-length protein, while its PX domain surprisingly interacted with two nuclear proteins, Std1p and Yra2p.
Finally, for Snx42p, Vam7p, and Vps17p we found different, but specific, interactions with both the full-length protein and the isolated domain. Interestingly, only Vam7p yielded interactions that were found with both the full-length protein and its PX domain.
Since we found eight interactions only with the PX domain but not the corresponding full-length protein, we conclude that full-length proteins often prevent interactions, possibly due to their fusion with the Gal4 domain fusion. Whatever the mechanistic reason may be, this indicates that we may have missed a significant number of interactions despite our comprehensive approach. Such false negatives may be prevented by more extensive usage of domains as baits.
The Nature of PX Protein Interactors
As expected from the role of many PX domain-containing proteins such as sorting nexins, the preys that we identified were enriched in proteins known to be involved in vesicular transport (36% of preys as opposed to 5% in the whole genome). Interestingly, a relatively large fraction of interactors were uncharacterized proteins (20 versus 30.5%). All other functional classes combined made up 44% of all preys (whole genome, 64%).
Similarly, when localization data was analyzed as opposed to functional annotation, the majority (49%) of preys has been localized to the secretory or endocytic pathway, that is, to the endoplasmic reticulum (ER), Golgi, endosomes, or the vacuole. Hence 53% of preys represented membrane or membrane-associated proteins, which was somewhat unexpected because membrane proteins are often said to resist detection in two-hybrid screens. Another unexpected finding was that Snx4p, Snx42p, and Mvp1p all interacted with a significant number of nuclear proteins.
No single domain or motif was common among our preys. However, some domains were significantly enriched. Four PX domain-containing proteins bind to another PX domain protein, resulting in four pairs: Vps17p-Vps5p, Vps17p-Snx4p, Snx4p-Snx42p, and Snx42p-Snx41p. However, because the PX domain-only baits do not reproduce these interactions, this reflects characteristics of other parts of these sorting nexinsparticularly their coiled-coil regions, which have been shown to drive homo- and hetero-oligomerization in other studies. This finding has been confirmed independently in a study of the Vps17p-Vps5p interaction (31).
A notable class of preys consists of the Yip1 family of proteins (32). Five out of the nine productive PX proteins (Ypt35p, Vam7p, Grd19p, Vps17p, Snx42p) interacted with at least one member of the Yip1 family, and these interactions were defined by the PX domain as assessed using PX domain-only baits and/or by in vitro interaction studies (see below).
Other domains that were found repeatedly among PX protein interactors were the SNARE domain (in Ypt35p interactions with Bet1p, Bos1p, and Pep12p), the Rab domain (Vps17p-Ypt53p, Grd19p-Ypt53p, Vam7p-Vps21p), and PH domains (Snx4p-Ask10p, Bem1p-Cdc24p). However, none of these interactions seems to involve the PX domain as only the full-length baits but not the PX-only baits detect those preys.
PX Domains Interact with Members of the Yip1 Family In Vitro
Given the surprisingly strong interactions of PX domains with the highly hydrophobic Yip1 family members in our two-hybrid screens, we wanted to verify these results by in vitro binding assays. All yeast PX domains that were positive in our two-hybrid screens were expressed as GST fusion proteins in E. coli and purified on glutathione-Sepharose beads. Note that the Mvp1p and Vam7p PX domains could not be tested because they were either unstable or insoluble. Yip1p, Yip4p, and Yif1p were translated and 35S-labeled in vitro and incubated with the GST-PX fusion proteins. Yip5p could not be tested because its in vitro translation repeatedly failed. Our in vitro binding assays clearly show that Yip1 family members bind specifically to certain PX domains (Fig. 3). The strongest interaction was shown for Ypt35-PX, which interacted strongly with Yif1p, and somewhat less with Yip1p and Yip4p. Vps17-PX showed a similar binding pattern, although the overall binding strength appears to be weaker. Snx4-PX only bound significantly to Yip4p. The PX domains from Snx42p preferred Yip1p over Yif1p, while the Vps5p PX domain appeared to bind most strongly to Yif1p. The interactions of isolated PX domains with Yip1 family members are summarized in Table III.
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By contrast, Grd19p interacted strongly with Yif1p, Yip1p, and Yip5p in a two-hybrid assay, but binding was barely detectable in vitro (Yip5 was not tested).
Finally, we observed one two-hybrid interaction, Snx42p-Yip1p, only with the full-length bait (but not the PX-only bait), although Snx42-PX interacted weakly with Yip1p and Yif1p in vitro.
Ypt35-PX Binds to Yip1 and Yif1 N Termini
Yip1 family members are extremely hydrophobic proteins with two to five predicted transmembrane domains (32). The only significant cytoplasmic part is an N-terminal domain of 100150 amino acids that is conserved among Yip proteins. We wondered if this domain interacts with PX domains. In vitro-translated Yif1p and Yip1p N termini indeed bound to GST-Ypt35-PX (Fig. 4). Binding assays with the remaining C-terminal half also showed some weak binding but were not conclusive (data not shown). Hence it remains unclear if the small cytoplasmic loops in the C termini of Yip1p and Yif1p contribute to PX binding.
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DISCUSSION |
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Yif1p and Yip1p have been reported to be localized to the Golgi (36, 37), and the two proteins have been postulated to be part of a complex (37). Recently, several studies showed that Yip1p/Yif1p proteins are necessary for COPII vesicle budding from the ER membrane (38) and for membrane fusion with the Golgi (39). Yip1p is thought to cycle between the ER and Golgi compartments (38).
It was therefore surprising that the strongest and most consistent PX domain-containing interactors of Yif1p/Yip1p were proteins that have been localized to the endocytic pathway. For example, Ypt35p is mainly found associated with endosomes (40), and Grd19p is localized to both the vacuolar membrane (40) and the cytosol (19), although it becomes associated with the prevacuolar compartment in vps27 null mutant cells (19). Vps17p also localizes to the endosome, as reported by a C-terminally tagged GFP fusion protein (40) as well as to the prevacuolar compartment (41). So do Snx42p and Snx4p, which have additionally been localized to the post-Golgi endosomes and the pre-autophagosomes (20). Snx42p is also found at a perivacuolar structure (35). And finally, Vam7p is usually seen in association with vacuolar membranes (42, 43).
There are three possible explanations for our finding that a group of ER/Golgi proteins (the Yip1 family) interact with endosomal/vacuolar proteins (the PX domain-containing proteins). First, a significant fraction of at least some PX proteins can be found in the cytoplasm, and could be recruited in small amounts to the ER membrane and act there during COPII vesicle formation. Although there is no direct evidence for this, while 5% of Vam7p associates with vacuolar membranes, the rest is soluble (43). Soluble and therefore diffusely stained cytoplasmic fractions of PX proteins could easily escape detection if there is at least one other membrane-related localization. In addition, sites of COPII vesicle formation do not seem to be visible easily by light microscopy and neither are the proteins involved in such highly dynamic membrane spots. Second, it remains likely that Yif1p/Yip1p proteins have other, yet unnoticed functions (independent of COPII vesicle formation) in post-Golgi sorting or endocytosis. This idea is supported by the requirement for the Yip1p/Yif1p-binding Rab GTPases in docking and fusion of transport vesicles, but not for budding (38). Furthermore, Sivars et al. 2003 (44) showed that Yip3, a mammalian Yip family member, can act as a GDI-displacement factor for the targeting of Rab GTPases, particularly Rab9. It may be interesting to determine the effect of PX proteins on the recruitment of GDFs, their activity, or some downstream event such as the recruitment of GEFs. Third and last, we cannot completely rule out the possibility that at least some of our interactions are experimental artifacts with no physiological relevance. However, the specificity, apparent strengths of the interactions, and confirmation by independent methods argue against this possibility.
Interestingly, Yif1p has been shown to localize to the vacuole under certain circumstances, for example in a Btn2 null mutant, which does not appear to disrupt vesicular transport per se (45). In addition, Yif1p was localized to the cytoplasm and may be free to diffuse to locations other than the ER (46).
Even if Yif1p and Yip1p do not have a role in endocytosis or post-Golgi sorting, such a function may have been taken over by Yip4p and Yip5p as their localization remains unclear. Yip5p localizes to the cytoplasm, as determined for a C-terminally tagged GFP fusion protein (40), while Yip4p does not appear to be localized in any study.
Nuclear PX Interactions
We were surprised to find that several PX domain-containing proteins showed preferential interactions with nuclear proteins. Although this was initially unexpected, it is clear that there are several relationships of inositol phosphates to nuclear functions (reviewed in Ref. 47). Mvp1p, a protein required for sorting proteins to the vacuole, interacted weakly with Std1p, a suppressor of a TBP deletion and thus likely a transcription factor (although its biochemical function is unknown). Mvp1p also interacted with Yra2p, a protein that is involved in mRNA export from the nucleus. Although the significance of both interactions is unclear, it is notable that both of these Mvp1p interactors have been found both in the nucleus as well as in the cytoplasm. Std1p has also been localized to the plasma membrane (48). Vps5p and Snx42p also have interactions with nuclear proteins of which Snx42ps interaction with Rrn10p is remarkable because it appears to be specific for the PX domain of Snx42p. Additional support for an Snx42p-Rrn10p interaction comes from the observation that Rrn10 null mutants are hypersensitive to wortmannin, an inhibitor of PI3 kinase (49).
The most remarkable example of nuclear protein interactions was seen with Snx4p, which has seven interactions with nuclear proteins. Two of these were highly reproducible, namely with Ecm11p and Sds3p. Ecm11p is an unusual protein in that its mutants exhibit phenotypes related to extracellular matrix functions such as zymolyase hypersensitivity. Nevertheless, the protein has been localized to the nucleus. Its molecular function remains obscure. The other nuclear Snx4p interactor, Sds3p, is a suppressor of defective silencing, i.e. it is involved in silencing the HMR mating cassette locus. Its precise biochemical activity is also unknown but, interestingly, it binds phosphoinositol 4-phosphate in vitro (50). Rrn10 and Sds3 are two of 38 genes whose deletion results in hypersensitivity to wortmannin but the mode of action remains to be determined.
In this article, we have shown that PX domains can interact with a number of different proteins. Most striking was their preference for membrane proteins of the Yip1 family. Given the much greater diversity of both Yip and PX proteins in vertebrate genomes, we hope that our findings will encourage further studies on these proteins and so improve our understanding of both their diversity, specificity, and, eventually, biological function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, July 19, 2004, DOI 10.1074/mcp.M400081-MCP200
1 The abbreviations used are: PI, phosphoinositide; PtdIns, phosphatidylinositol; PX, phox homology domain; SH3, Src homology 3; ER, endoplasmic reticulum.
* This work has been funded by the DFG (Ue 50/21) and the Helmholtz Association.
S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material.
To whom correspondence should be addressed: Institut für Genetik, Forschungszentrum Karlsruhe, Box 3640, D-76021 Karlsruhe, Germany. Tel.: 49-7247-82-6103; Fax: 49-7247-82-3354; E-mail: peter.uetz{at}itg.fzk.de
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REFERENCES |
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