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. PalBΔMIT, 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 PalBΔMIT. 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.
Highlights
The multivesicular body (MVB)3 pathway plays a central role in the delivery of membrane proteins reaching the endosomal system to the lumen of the vacuole/lysosome [1]
Fax: 34915360432; E-mail: penalva@cib.csic.es. 3 The abbreviations used are: MVB, multivesicular body; 2H, two-hybrid; ESCRT, endosomal sorting complex required for transport; GST, glutathione S-transferase; MIM, MIT domain-interacting motif; MIT, microtubule interacting and trafficking; TMD, transmembrane domain; GFP, green fluorescent protein; HA, hemagglutinin; UTR, untranslated region
The ultimate consequence of MVB sorting for an endocytosed membrane cargo is exposure to the late endosome/lysosome lumenal hydrolases, which inevitably ends with proteolytic degradation
Summary
A. nidulans—palB gene replacements were carried out using MAD1764 (pyrG89 pyroA4 inoB2 ⌬nkuA::bar pacC900 ⌬palB::pyroAAf), a ⌬palB strain in which the complete coding region of palB had been substituted by pyroAAf. GST fusion proteins were expressed in E. coli DH1 for 24 h at 20 °C after induction with isopropyl-1-thio-D-galactopyranoside and purified from lysates clarified by centrifugation for 20 min at 13,000 rpm and 4 °C in a Sorvall SS-34 rotor as described [51]. GST baits were mixed with 1 mg of total soluble protein extract from palB800 or palB801 strains in BOA buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 0.05% (v/v) Triton X-100, and Roche’s protease inhibitor mixture), using 0.8-ml Handee Spin columns (Pierce), and rotated at 4 °C in the presence of 25 l of glutathione-Sepharose (Amersham Biosciences). ZZ-PalBMIT was purified from cells lysed in the French Press in a buffer containing 20 mM Tris-HCl, pH 7.9, 300 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, 1 mM PFA-block, and Roche Applied Science’s EDTA-free protease inhibitor mixture. These were combined with peroxidasecoupled sheep anti-mouse IgG (Amersham, 1/4,000), goat antirat IgMϩIgG (goat, 1/4,000) or donkey anti-rabbit IgG as secondary antibodies, which were reacted with ECL (Amersham Biosciences)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
