Bub3-Bub1 Binding to Spc7/KNL1 Toggles the Spindle Checkpoint Switch by Licensing the Interaction of Bub1 with Mad1-Mad2.

The spindle assembly checkpoint (SAC) ensures that sister chromatids do not separate until all chromosomes are attached to spindle microtubules and bi-oriented. Spindle checkpoint proteins, including Mad1, Mad2, Mad3 (BubR1), Bub1, Bub3, and Mph1 (Mps1), are recruited to unattached and/or tensionless kinetochores. SAC activation catalyzes the conversion of soluble Mad2 (O-Mad2) into a form (C-Mad2) that binds Cdc20, BubR1, and Bub3 to form the mitotic checkpoint complex (MCC), a potent inhibitor of the anaphase-promoting complex (APC/C).

Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal compounds.

The pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7-TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events.

Rescue of Aspergillus nidulans severely debilitating null mutations in ESCRT-0, I, II and III genes by inactivation of a salt-tolerance pathway allows examination of ESCRT gene roles in pH signalling.

The Aspergillus pal pathway hijacks ESCRT proteins into ambient pH signalling complexes. We show that components of ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III are nearly essential for growth, precluding assessment of null mutants for pH signalling or trafficking. This severely debilitating effect is rescued by loss-of-function mutations in two cation tolerance genes, one of which, sltA, encodes a transcription factor whose inactivation promotes hypervacuolation.

Endocytic machinery protein SlaB is dispensable for polarity establishment but necessary for polarity maintenance in hyphal tip cells of Aspergillus nidulans.

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence.

Receptor-independent Ambient pH signaling by ubiquitin attachment to fungal arrestin-like PalF.

The seven-transmembrane receptor PalH and its coupled, positive-acting arrestin-like protein PalF are key components of a molecular sensor that in Aspergillus nidulans and other ascomycete fungi mediates activation of an intracellular signaling cascade by alkaline ambient pH. PalF is ubiquitinated in an alkaline pH- and PalH-dependent manner. We show here that PalF assists the plasma membrane localization of PalH and that PalF overexpression slightly hypersensitizes the pathway to alkaline pH but does not bypass the need for the ambient pH signal receptor in signaling.

Characterization of Aspergillus nidulans DidB Did2, a non-essential component of the multivesicular body pathway.

ESCRT-III heteropolymers mediate membrane protein cargo sorting into multivesicular endosomes for subsequent vacuolar degradation. We studied the localization of largely uncharacterized Aspergillus nidulans ESCRT-III using its key structural component Vps32 and the 'associated' component DidB(Did2). Vps32-GFP localizes to motile early endosomes as reported, but predominates in aggregates often associated with vacuoles due to inability to dissociate from endosomes.

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

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.

Establishment of the ambient pH signaling complex in Aspergillus nidulans: PalI assists plasma membrane localization of PalH.

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH.

Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC.

The 72-kDa zinc finger transcription factor PacC, distantly related to Ci/Gli developmental regulators, undergoes two-step proteolytic processing in response to alkaline ambient pH. "Signaling protease" cleavage of PacC(72) removes a processing-inhibitory C-terminal domain, making its truncated PacC(53) product accessible to a second "processing" protease, yielding PacC(27). Features of the processing proteolysis suggested the proteasome as a candidate protease.

PalC, one of two Bro1 domain proteins in the fungal pH signalling pathway, localizes to cortical structures and binds Vps32.

PalC, distantly related to Saccharomyces cerevisiae peripheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulans pH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH.

Further characterization of the signaling proteolysis step in the Aspergillus nidulans pH signal transduction pathway.

The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved "signaling protease box" in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB.