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. 2009 Feb 13;284(7):4404-12.
doi: 10.1074/jbc.M808645200. Epub 2008 Dec 3.

Physiological involvement in pH signaling of Vps24-mediated recruitment of Aspergillus PalB cysteine protease to ESCRT-III

Olga Rodríguez-Galán  1 , Antonio Galindo, América Hervás-Aguilar, Herbert N Arst Jr, Miguel A Peñalva
Affiliations

Physiological involvement in pH signaling of Vps24-mediated recruitment of Aspergillus PalB cysteine protease to ESCRT-III

Olga Rodríguez-Galán et al. J Biol Chem. .

Abstract

Activation of the Aspergillus nidulans transcription factor PacC, which mediates ambient pH regulation of gene expression and is recruited to ESCRT-III by the Vps32-interacting scaffold PalA, involves its ambient pH-dependent C-terminal proteolysis. This reaction is almost certainly catalyzed by the PalB calpain-like protease. Here we show that PalB associates with membranes and interacts specifically and directly with ESCRT-III Vps24. The PalB N-terminal MIT domain and the Vps24 C-terminal MIM motif are necessary and sufficient for this interaction. PalB(DeltaMIT), a mutant PalB lacking the MIT domain is inefficiently recruited to membranes and impaired in PacC proteolytic processing. Notably, membrane recruitment is promoted and PacC processing largely restored by covalent attachment of Vps24 to mutant PalB(DeltaMIT). This is the first reported evidence that calpain-like recruitment to ESCRT-III lattices plays a physiological role. It unambiguously positions the calpain-like protease PalB within the ESCRT-III-associated pH signaling complex, underlines the positive role of ESCRT-III in ambient pH signal transduction, and suggests a possible mechanism for PalB activation.

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Figures

FIGURE 1.
FIGURE 1.
PalB interacts with Vps24 of ESCRT-III. A, schemes of the domain organizations of PalB and of Vps24; B and C, two-hybrid analyses: the indicated GAL4 DNA binding domain (pGBKT7) and activation domain (pACT2AD) constructs were tested in S. cerevisiae Y187, using quantitative β-galactosidase assays. Data represent average values of five clones for each combination. Bars indicate standard errors. Φ indicates empty pGBKT7 or pACT2 vectors. D, GST-Vps24 pull-down assays of A. nidulans extracts expressing physiological levels of PalB or PalBΔMIT, as indicated. Input lanes contained 2% of the pulled-down material. PalB was detected by α-HA Western blotting.
FIGURE 2.
FIGURE 2.
PalB binds directly to Vps24. Key residues in the Vps24 MIM domain are essential for PalB binding. A, autoradiographs of GST pull-down assays with the indicated A. nidulans GST fusion proteins and 35S-labeled PalA and PalB preys synthesized in vitro using a coupled transcription/translation system. Input lanes contained 2% of the material used in the pull-downs. Note that GST-DidB does not pull-down PalB under these conditions. B, amino acid sequence comparison of the MIM motifs of fungal Vps24 and Vps2 proteins and their respective CHMP3 and CHMP2A human orthologues is shown on the left. Shaded residues correspond to the consensus MIM motif recognizing the Vps4 MIT domain (38, 42). AnVps24 Arg-222 and Leu-223 correspond to positions 0 and 1 in the consensus motif. C, GST pull-down assays using wild-type or mutant (R222D and L223D substituted) A. nidulans GST::Vps24 fusion proteins as baits and 35S-labeled wild-type PalB as prey.
FIGURE 3.
FIGURE 3.
A. nidulans vps24 is a virtually essential gene. A, scheme of a conidiophore of an A. nidulans heterokaryotic strain carrying untransformed vps24+, pyrimidine-requiring haploid nuclei (blue) and transformed vps24Δ, pyrimidine-independent nuclei (red) (see also the color scheme in B). During conidiophore development, individual nuclei segregate into conidia, thereby resolving the heterokaryotic situation. Thus, heterokaryotic pyrG- strains carrying nuclei where an essential gene has been replaced by a pyrG+ allele can be propagated from conidiospores in the presence but not in the absence of pyrimidines, as the auxotrophic conidia corresponding to the untransformed nuclei required uracil supplementation for growth. Transformants 1 through 5, 7, and 8 are heterokaryotic, whereas transformant 6 is a spontaneous heterozygous diploid or aneuploid for vps24. B, Southern blot analysis of the transformants with indication of diagnostic bands. Note that, in heterokaryotic transformants 1–5, the intensities of the vps24Δ bands relative to the vps24+ band are lower than in the diploid/aneuploid transformant 6, indicating that the heterokaryons contain fewer vps24Δ than vps24+ nuclei.
FIGURE 4.
FIGURE 4.
Deletion of the PalB MIT domain impairs its association with membranes and the processing of PacC. A, MIT domain-mediated association of PalB with membrane fractions. Wild-type palB800 and mutant palB801ΔMIT protoplast lysates were fractionated into 13,000 × g insoluble (P13) and 100,000 × g insoluble (P100) and soluble (S100) fractions, which were analyzed by Western blotting. B, palB801ΔMIT results in weak loss-of-function in diagnostic plate tests of pH signaling (16, 74). palB800 is phenotypically wild-type (data not shown). In contrast, palB801ΔMIT confers some neomycin resistance (Neo), weakly impairs growth on alkaline pH (OH) and molybdate (Mo) plates, and decreases tolerance of LiCl (Li), indicating that deletion of the MIT domain results in weak loss-of-function; SC is synthetic complete medium without any addition. C, similar stability of PalB and PalBΔMIT in pH shift experiments. PalB was detected using α-HA antibody. Similar loading in the different lanes was determined using β-actin. palB800 and palB801ΔMIT cells cultured at acidic ambient pH were shifted to alkaline pH. Cell extracts were analyzed by Western blotting. D, palB801ΔMIT impairs PacC processing in pH shift experiments. Wild-type palB800 and null palBΔ controls are shown. The three forms of PacC are indicated.
FIGURE 5.
FIGURE 5.
Attachment of Vps24 to the N terminus of PalBΔMIT significantly restores function. A, transgene expressing a Vps24:: PalBΔMIT fusion protein was constructed by gene replacement as described in supplemental Fig. S1. While PalBΔMIT predominates in the soluble fraction, N-terminal attachment of Vps24 redirects the protein to P13 membranes. B, attachment of Vps24 to the N terminus of PalBΔMIT results in a marked increase in the two-step processing of PacC, such that PacC53 becomes clearly detectable in a pH shift experiment.
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