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Previous Article | Next Article 
Molecular and Cellular Biology, March 2003, p. 1647-1655, Vol. 23, No. 5
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.5.1647-1655.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Joan Tilburn,2 Herbert N. Arst, Jr.,2 and Miguel A. Peñalva1
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Madrid 28006, Spain,1 Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, London W12 0NN, United Kingdom2
Received 4 October 2002/ Returned for modification 21 November 2002/ Accepted 2 December 2002
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ABSTRACT |
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INTRODUCTION |
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Ambient pH signaling occurs under alkaline conditions. In such circumstances, pH signal transduction enables the activation of the otherwise transcriptionally inactive 674-residue PacC translation product by a two-step proteolysis mechanism which can be compared to regulated intramembrane proteolysis. In the first step, which is crucially regulated by ambient pH, the 72-kDa PacC translation product (PacC72) is converted to a 53-kDa intermediate (PacC53) lacking the 
180 C-terminal residues (see Fig. 1A). This step is catalyzed by the signaling protease, likely to be PalB. In a second, pH-independent step, this (committed) intermediate is converted to the 27-kDa processed product (PacC27) containing the 250 N-terminal residues by an as yet unidentified processing protease (see Fig. 1A) (8, 10, 25, 31). The crucial feature of this regulatory mechanism, which involves changes in PacC nucleocytoplasmic distribution (24), is that PacC72 is protected from activating proteolysis under inappropriate circumstances (i.e., acidic ambient pH and absence of pal signaling) by adopting a closed conformation, which prevents accessibility to the processing protease. This model is supported by mutational analysis of pacC (8, 10, 25, 40). Thus, alkalinity-mimicking pacCc C-terminally truncating mutations (mimicking the signaling protease cleavage) or certain amino acid substitutions disrupt the closed conformation, leading to pH-independent activation of PacC, whereas a subclass of acidity-mimicking pacC+/- mutations prevent the signaling cleavage, resulting in closed PacC72 at any ambient pH.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyrate as a nitrogen source in glucose minimal medium, all other novel pacC alleles (described in Table 1) were constructed in vitro by PCR using mutagenic primers and introduced into A. nidulans by homologous gene replacement of a 
pacC::pyr4 allele as described previously (8). Candidate strains were confirmed by Southern analysis.
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Two-hybrid analysis.
The Saccharomyces cerevisiae strain used for two-hybrid studies was CTY10.5d (Mata ade2-101 his3-200 leu2-1 trp1-901gal4 gal80 URA3::lexAop-lacZ). Standard genetic methods were used. Yeast cells were grown in synthetic dextrose minimal medium (SD) lacking appropriate supplements, to maintain selection for plasmids (36). For ß-galactosidase assays, transformants were patched onto selective SD medium and grown for 2 days at 30°C. Filter lift assays for blue color were performed as described previously (47), and the cultures were developed for 1 h. For quantitative assays, four different transformants were grown to mid-log phase in selective SD medium. ß-Galactosidase activity was assayed in permeabilized cells and expressed in Miller units (23). Yeast protein extracts for immunoblot analysis were prepared as described previously (42) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide) followed by immunoblotting with a monoclonal anti-HA antibody (Roche).
Plasmids. The plasmids used in this study are listed in Table 2. pLexA-PalA, pGAD-PalA, and pGST-PalA were constructed by inserting a PCR fragment containing the PalA coding sequence in the polylinker site of pLexA(1-202)+PL, pACTII, and pGEX-2T, respectively. Constructs encoding PacC fusion proteins to the Gal4 activating domain (GAD) were derived from pACTII by inserting the corresponding PacC fragments into the BamHI site of the polylinker. Site-directed pacC mutations were obtained by PCR-directed sequence modification and first introduced into pSpacC, a pBS-SK(+) derivative containing the pacC gene (8). pGST-PacC(529-678), pGST-PacC(169-410), pHis-PacC, and pHis-PacCL340S have been described previously (10). pHis-PacCY455D-Y662N was constructed by replacing a fragment from pHis-PacC with a fragment from a pSpacC derivative containing the corresponding mutations. To construct pLexA-SVMYPTLRGL and mutant derivatives, we inserted a double-stranded oligonucleotide with the corresponding coding sequence followed by a stop codon in the polylinker of pLexA(1-202)+PL. pLexA-Vps32 is a pLexA(1-202)+PL derivative containing the VPS32 coding sequence (our unpublished results) amplified by PCR from A. nidulans genomic DNA. The pACTII derivative, pGAD-AIP1/Alix, was constructed by PCR with a human AIP1/Alix clone (IMAGE 5284284) as template.
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Glutathione S-transferase (GST)-PalA, GST-PacC(529-678), GST-PacC(169-410), and GST were expressed from Escherichia coli DH1 transformed with the corresponding plasmids (Table 2). Synthesis of these proteins was induced at 37°C by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) to 50-ml cultures, which were incubated for an additional 3 h. Bacterial pellets were resuspended in 7 ml of STE buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA). After addition of 1% (vol/vol) Triton X-100, 5 mM dithiothreitol, and Complete protease inhibitor cocktail (Roche), the cells were lysed in a French press. Extracts were cleared by centrifugation and incubated (1-ml portions) with glutathione-Sepharose 4B beads (Pharmacia) at 4°C for 1.5 h. After being washed six times with 1 ml of STE buffer plus 1% (vol/vol) Triton X-100, beads with bound proteins were split into equal portions. In vitro-synthesized wild-type and mutant PacC proteins (5 to 7 µl) were added to the beads and allowed to bind at 4°C for 1 h in 500 µl of STE buffer with 1% Triton X-100. After five washes with STE-1% Triton X-100 and a further wash with STE buffer, the beads were boiled in sample buffer and proteins were separated by SDS-PAGE (10% polyacrylamide). Bound proteins were detected by autoradiography (labeled preys) or Coomassie staining (baits).
EMSA and immunoblot analysis of A. nidulans protein extracts. A. nidulans protein extracts were prepared from lyophilized mycelium as described previously (8). Electrophoretic mobility shift assays (EMSA) were performed by the method of Orejas et al. (31), using a32P-labeled double-stranded oligonucleotide containing the IpnA2 PacC binding sequence and 5 µg of A. nidulans protein extracts. Electrophoresis was performed in 4% (wt/vol) polyacrylamide gels. For Western analysis, 50 µg of A. nidulans protein extract was resolved by SDS-PAGE (10 or 12% polyacrylamide) and PacC was detected using a rat anti-PacC(5-265) polyclonal antiserum (1/2,000) (25) and a peroxidase-conjugated goat anti-rat secondary antibody (1/4,000) (Southern Biotechnology). Peroxidase activity was detected by enhanced chemiluminescence with ECL reagents (Amersham).
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RESULTS |
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Region D contains a YPXL/I motif which is repeated upstream of the signaling protease cleavage site.
Multiple sequence alignment of fungal PacC proteins and their Rim101p yeast homologues did not reveal large blocks of sequence conservation outside of the DNA binding domain. However, a closer inspection of the alignment of the C-terminal moieties in PacC/Rim101p proteins revealed two short, completely conserved motifs (consensus YPXL/I) at positions 455 and 662 of A. nidulans PacC, which are present in all PacC/Rim101p family members (Fig. 2). The more C-terminal of these motifs (Box 2) is located very close to the C termini of the proteins (Fig. 2), within region D of A. nidulans PacC defined above. The more N-terminal motif (Box 1) overlaps in all cases with an SH3 binding motif, PXXP (either YPXL/IP in filamentous fungi or PXYPXL/I in yeasts [Fig. 2]). In A. nidulans PacC, Box 1 is placed 40 residues upstream of the signaling protease cleavage site, located at residue 500. Of note, Candida albicans Rim101p contains a duplication of Box 1 (Fig. 2).
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PalA binding to YPXL/I motifs in PacC is direct and independent of the PacC conformation. To provide corroborating evidence that PalA and PacC interact and to show that this interaction is direct, we assayed the binding of in vitro-synthesized PacC to a purified, bacterially expressed GST-PalA fusion protein. GST-PalA or GST (Fig. 4A) was immobilized on glutathione-Sepharose beads and incubated with [35S]PacC, synthesized in vitro using a coupled transcription-translation system. Whereas no binding was detected with GST alone, PacC was retained by GST-PalA beads (Fig. 4B, lanes 4 and 7). This binding was prevented when a double-mutant (Y455D-Y662N) PacC protein was used (lane 8), demonstrating its dependence on the integrity of the YPXL/I motifs. Together, these results indicate that PalA and PacC interact directly via the YPXL/I motifs in PacC.
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The two YPXL/I motifs in PacC play a physiological role in vivo. To address the physiological role of the YPXL/I motif-mediated interactions between PacC and PalA, we introduced by gene replacement the pacC mutations leading to Tyr455Asp and Tyr662Asn substitutions, alleles pacC+/-211 and pacC+/-212, respectively.
The Tyr455Asp substitution (pacC+/-211) results in a stringent loss-of-function, acidity-mimicking phenotype. A pacC+/-211 mutant does not grow at alkaline pH and is hypersensitive to molybdate, hyperresistant to neomycin, and strongly derepressed for extracellular acid phosphatase at pH 6.8 (Fig. 5). Further proof of the critical involvement of Tyr455 in PacC function is provided by the classically selected, stringently acidity-mimicking pacC+/-207 mutation resulting in Tyr455Asn. pacC+/-207 is phenotypically indistinguishable from pacC+/-211 (data not shown). These data indicated that Y455PXL/I-mediated recognition of PacC by PalA is a major requirement for reception of the pH signal. Of note, the acidity mimicry of pacC+/-211 or pacC+/-207 is only slightly less extreme than that resulting from the pacC+/-20205 loss-of-function mutation (Fig. 5 and data not shown). pacC+/-20205, which prevents the signaling cleavage step (8), phenotypically resembles null mutations in the palA, palB, palC, palF, and palH genes, inactivating the pH signal transduction pathway (25).
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The YPXL/I motifs are required for pH-dependent proteolytic activation of PacC. Transduction of the ambient pH signal leads to cleavage of transcriptionally inactive PacC72 by the signaling protease (presumably PalB) to yield PacC53, the substrate of the processing protease. Using EMSA and Western analyses of mycelial extracts, we examined the involvement of the PacC YPXL/I motifs in pH signal reception. Mycelia of the wild-type and pacC mutant strains were grown under acidic conditions and shifted to alkaline conditions for 45 min. Wild-type PacC72 is fully converted to the processing intermediate (PacC53) and processed form (PacC27) within 45 min after the shift (8), as determined both by EMSA (Fig. 6A, lanes 1 and 2; note that the PacC53-DNA complex shows reduced mobility compared to the PacC72 complex due to conformational differences) or by Western blot analysis (Fig. 6B, lanes 1 and 2). The pacC+/-211 (Tyr455Asp) product was severely impaired in the signaling protease step and therefore was poorly processed (Fig. 6, lanes 7 and 8). These and the above results unambiguously demonstrate that PalA binding to the PacC Box 1 Y455PXL/I motif is required for the signaling cleavage step. In contrast, and as expected from its much weaker acidity-mimicking phenotype, pacC+/-212 (Tyr662Asn), inactivating the more C-terminal Box 2 Y662PXL/I motif, was only partially deficient in pH signal reception (Fig. 6, lanes 3 and 4). pacC+/-212 appeared to impair the signaling cleavage to a certain extent, since it resembled pacC+/-211 in leading to a marked accumulation of PacC72 but, in contrast, did not prevent the formation of the intermediate (lanes 3 and 4). In agreement with phenotypic tests, the effect of pacC+/-213, truncating PacC immediately upstream of the Y662PXL/I motif, is indistinguishable from that of pacC+/-212 (lanes 5 and 6), demonstrating that the Tyr662Asn substitution fully inactivates the motif. These and the above results indicate that the Box 1 and Box 2 motifs are physiologically involved in the role of PalA in pH signal transduction.
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PalA homologues are widespread in the eukaryotic world (27, 46), including vertebrates, where the PalA family members include the apoptosis-related protein AIP1/Alix (26, 44). This raised the possibility that the ability of PalA to interact with the YPXL/I motif might be conserved in AIP1/Alix proteins. To address this point, we tested the human AIP1/Alix protein (GenBank accession no. AF151793 [45]; 26 % identity to PalA over 798 residues) in a two-hybrid assay. It interacted strongly with the Y662PXL/I-containing decapeptide, with a sequence specificity indistinguishable from that of PalA, as determined by Ala scanning mutagenesis (Fig. 7). Thus, the ability of PalA and its homologues to recognize the YPXL/I motif is conserved from fungi to humans.
Conserved two-hybrid interaction between PalA/AIP1/Alix family members and Vps32, a class E Vps protein.
Vacuolar-protein sorting (Vps) proteins are largely conserved from fungi to humans (18). For example, homologues of yeast Vps32p showing 38% amino acid sequence identity are found in both the A. nidulans and human genomes (our unpublished results). In view of biochemical and genetic evidence which indicates an interaction between yeast PalA family members and Vps32 (reviewed in reference 33) (see Discussion; Fig. 8), we tested by two-hybrid analysis whether this interaction is conserved in A. nidulans. Figure 7 shows that PalA strongly interacts with the putative A. nidulans Vps32. Remarkably, this ability to interact with A. nidulans Vps32 is conserved in human AIP1/Alix (Fig. 7). Since fungal and human Vps32p homologues do not contain YPXL/I motifs, these two-hybrid assay results, together with yeast biochemical and genetic data (14, 15, 41), suggest that the ability to interact with this class E Vps protein is an additional feature of PalA/AlP1/Alix family members.
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DISCUSSION |
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The ambient pH-dependent step in the proteolytic activation of the fungal PacC zinc finger protein is the cleavage of PacC72 (the translation product) to yield the PacC53 intermediate, which is committed to proteolytic activation (Fig. 1A). This conversion of PacC72 to PacC53 has been denoted the signaling protease step and is probably mediated by the PalB calpain-like cysteine protease, a homologue of human calpain 7. By exploiting the ease with which A. nidulans can be manipulated genetically, we show that PalA binding to the two YPXL/I motifs in PacC is required for the action of the signaling protease. Although the strength of the interaction between each site and PalA appears to be rather similar, as determined by a two-hybrid assay (Fig. 3), the contributions of each YPXL/I motif to pH signal reception appear to be markedly different. Substitution of the critical Tyr residue in the more N-terminal motif severely impairs the signaling protease step and therefore leads to a tight pacC loss-of-function phenotype. In contrast, deletion or tyrosine substitution in the more C-terminal motif has a less pronounced effect and leads to a weak loss-of-function phenotype. The molecular basis for the differing physiological contributions of the two YPXL/I motifs to ambient pH signal-dependent proteolytic activation is unknown, although we note the presence of an SH3 binding site overlapping the most N-terminal motif (Box 1 in Fig. 2) contained in all members of the PacC/Rim101p family of zinc finger proteins. This might suggest that PalA binding to this site antagonizes the binding of a negatively acting SH3 domain-containing protein.
Although the pH signal transduction pathway is widespread in both yeast and filamentous ascomycetes (33), proteolytic activation of PacC/Rim101p proteins has been demonstrated only in A. nidulans (31) and S. cerevisiae (20). However, the strict conservation of the two YPXL/I motifs in all members of the family (Fig. 2), together with extant mutational evidence (reviewed in reference 33), strongly suggests that all these proteins undergo at least one common proteolytic activation step in which protein-protein recognition through YPXL/I motifs plays a crucial role. In agreement, two YPXL/I motifs in the S. cerevisiae PacC homologue, Rim101p, are located in two regions of this protein that were previously found to interact with Rim20p, the PalA homologue (46).
PalA is able to bind YPXL/I motifs in PacC72, i.e., the translation product in the closed conformation. This would be consistent with PalA playing a role in recruiting the signaling protease, in disrupting the intramolecular interactions maintaining the closed conformation, or both. The calpain-like cysteine protease PalB is likely to be the signaling protease (8). In yeast, Rim20p and Rim13p, the respective PalA and PalB orthologues, interact with Snf7p/Vps32p, an endosome-associated class E Vps protein (15) (see below), which led to the suggestion that Rim20p would recruit Rim13p to its substrate Rim101p (46). Functional interaction between PalA homologues and cysteine proteases extends beyond the fungal world. For example, mammalian AIP1/Alix interacts with ALG-2, a member of the calpain small-subunit superfamily (reviewed in reference 38), which is involved in apoptosis and is a potential calpain regulatory subunit (21, 26, 43, 44). In view of the strength and complete conservation in amino acid sequence specificity of the interaction between AIP1/Alix or PalA and the YPXL/I motif, we hypothesize that AIP1/Alix might recruit YPXL/I-containing substrates to the putative ALG-2-associated cysteine protease.
Two PalA homologues, Rim20p and Bro1p, are present in the yeast proteome, but only Rim20p plays a role in pH regulation (29, 46). Data from several laboratories strongly suggest that these proteins physically interact with certain class E Vps protein complexes acting at the cytosolic side of endosomes to mediate the sorting of transmembrane proteins into the multivesicular body (MVB) pathway. In genome-wide two-hybrid screens, Rim20p interacts with two class E Vps proteins, Vps32p and Vps4p (15, 17, 41) (Fig. 8). Snf7p/Vps32p is a component of ESCRT-III (endosomal sorting complex required for transport III) acting at a late stage in the sorting of endosomal cargo into the MVB pathway (1, 2). Vps4p is an AAA ATPase which regulates the dissociation of Snf7p/Vps32p-containing ESCRT-III complexes from the endosomal membrane pathway (1). In agreement with these two-hybrid assay results, large-scale in vivo analysis of yeast protein complexes (14) revealed Bro1p in physical association with Vps4p and Vps32p (Fig. 8). Indeed, BRO1 is allelic to Vps31, another member of the class E vps family (39). We show here that the interactions of Bro1p and Rim20p with Vps32p are conserved in fungal PalA and human AIP1/Alix, suggesting that proteins of the PalA family physically connect YPXL/I-containing proteins to MVB pathway sorting complexes.
The YXX
endocytotic motif described in higher eukaryotes (where is a bulky hydrophobic residue) (30) would appear to resemble the YPXL/I PalA/AIP1/Alix binding motif. However, not only are the consensus sequences of these two motifs different (3) (Fig. 7; see above) but also the motifs are functionally unrelated. Thus, while the YPXL/I motif appears to be connected with late endocytosis/MVB pathway class E protein complexes, YXX motifs recruit the early endocytotic machinery to plasma membrane proteins by interacting with the µ2 subunit of the AP2 adapter complex of clathrin-coated pits (30).
In contrast, the sequence specificity of the PalA/AIP1/Alix binding motif (Fig. 7) is indistinguishable from that of the YPDL late-domain motif identified in the GAG p9 protein of the equine infectious anemia virus (EIAV), in which replacement of Tyr, Pro, or Leu by Ala abolishes function (35). Late-domain motifs are short sequence motifs present in GAG p9 proteins of retroviruses which are required for efficient release of budding virions from the plasma membrane of infected cells. These GAG p9 late-domain motifs include, in addition to EIAV YPDL, the human immunodeficiency virus type 1 PTAP and the Rous sarcoma virus PPPY motifs (reviewed in reference 34). Despite their different amino acid sequences, the three GAG p9 late-domain motifs are functionally interchangeable, suggesting that they work by recruiting different subunits of a multiprotein complex involved in budding of all three viral particles (19, 32, 35).
Recent work (13) demonstrated that PTAP and PPPY motif function is dependent on (mammalian) Vps4, the key AAA ATPase required for disassembly of ESCRT-III complexes from the endosomal membrane. In addition, human immunodeficiency virus type 1 PTAP motif function is mediated through interaction with mammalian Tsg101/Vps23 (5, 13), an ESCRT-I complex protein also involved in the MVB pathway (1, 2, 16). These data point to a class E protein complex as the multiprotein machinery mediating GAG p9 late-motif function. Therefore, the likelihood that the EIAV GAG p9 motif and the YPXL/I PalA/AIP1/Alix binding motif not only share their consensus sequences but also share the ability to interact with the class E Vps machinery is very suggestive and may help us understand the thus far elusive function of human AIP1/Alix.
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ACKNOWLEDGMENTS |
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We thank the CICYT, the BBSRC, and the EU (through grants BIO2000-920 [to M.A.P.], 60/P11494 [to H.N.A.], and QLK3-CT-1999-00729 [to M.A.P. and H.N.A.]) for support. O.V. held an EMBO long-term fellowship and is currently supported by the Ramón y Cajal Program (MCyT; Spain). L.R. held a BBSRC studentship.
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FOOTNOTES |
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Present address: Division of Reproductive and Child Health, Medical and Molecular Genetics, The Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom.
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