Rim20 localizes to endosomes under alkaline pH conditions and is essential for proteolytic activation of the Rim101 transcription factor. Prior studies with S. cerevisiae vps4
Δ mutants suggested that Rim20-endosome association may be the limiting step for activation of Rim101 (4
). This model was based on the finding that the vps4
Δ defect promoted both Rim20-endosome association and partial Rim101 activation under a variety of conditions that were restrictive in VPS4
mutant strains. We report here that a bro1
Δ mutant strain separates the two outcomes, permitting Rim20-endosome association under otherwise restrictive conditions but not Rim101 activation. Our observations together indicate that there must be an additional level of pH- and/or Rim101 pathway-dependent control over the Rim20-Rim13-Rim101 complex that leads to Rim101 cleavage.
Our experiments suggest that Bro1 domain proteins, expressed at their natural levels, compete for endosome association. These findings are consistent with the overall similarity of the Bro1 domains of Bro1 and Rim20 and with the finding that many alanine scan mutations in SNF7
impair both Bro1- and Rim20-related functions (22
). In fact, Rim20-Bro1 competition may help to explain why snf7
mutants whose protein products have only a twofold reduced ability to bind Bro1 in vitro
have endocytic trafficking defects as severe as those of a snf7
Δ null mutant strain (21
). Similarly, competition by Bro1 may contribute to the Rim101 pathway-specific phenotypes of the alanine scan snf7
). However, we did not see functional consequences of such competition with WT Snf7 in experiments reported here. It is likely that simple replacement of Bro1 with Rim20 does not open up sites on the endosomes for other essential upstream components of the Rim101 pathway to assemble. In fact, it has been shown recently that Snf7 binding to both ESCRT proteins and Rim proteins is much stronger under alkaline growth conditions (21
), which might explain why artificial Rim20 localization does not recapitulate other changes in the cell upon a shift to alkaline pH. From these results, it seems possible that the consequences of Bro1 domain competition may have been minimized through evolutionary selection.
Interestingly, when Rim20 substitutes for Bro1 at endosomal sites, it assumes the regulatory requirements for Bro1-endosome association. Specifically, under acidic conditions in the bro1
Δ mutant background, Rim20-GFP foci are independent of upstream Rim101 pathway components and become dependent upon Vps27. This observation fits well with our mechanistic understanding of ESCRT-I recruitment to endosomes. Vps27 promotes ESCRT-I recruitment, presumably in response to the monoubiquitination signal that marks plasma membrane proteins for endocytosis (17
). Recent results from Vincent and colleagues suggest that the Rim101 pathway upstream components, via Rim8, may function analogously in alkaline pH-dependent ESCRT-I recruitment (7
). Thus, it seems reasonable that most endosomal ESCRT under acidic growth conditions depends upon Vps27, because Rim8 and other upstream Rim101 pathway components are inactive.
Using double-mutant analysis in the absence of BRO1, we find upregulation of Rim20 localization but no suppression of Rim101 processing defects, which is unlike that seen in vps4 mutants. One idea to reconcile these findings is that trafficking of Rim101 pathway components necessary for processing complex formation may differ between the two mutant backgrounds. This would imply that a trafficking defect exists in the absence of Bro1 but not in the absence of Vps4, yet no trafficking evidence supports this model. We favor a simpler kinetic model, based on a functional differentiation between Vps4 and Bro1. Vps4 is an enzyme critical for endosomal protein disassociation, while Bro1 is a structural protein whose endosome disassociation is controlled by Vps4. In the absence of Vps4, significant endosome-protein capture and sequestration occur, including Rim20 and Rim13—the protease that cleaves Rim101—along with Rim101 itself and possibly other components. In contrast, Bro1 is a structural protein, and loss of Bro1 likely only affects the endosome association status of Rim20, with little effect on any additional proteins. Consistent with a carefully calibrated kinetic mechanism, as mentioned above, Rim20 foci are most abundant immediately following pH shock and recede within 10 min, suggesting an acute regulated time period of processing complex formation.
Previously, it was shown that in the absence of Bro1, Rim101 is processed efficiently but there is still a defect in transcriptional repression by Rim101 (20
). Repression was assayed with a site that required the activity of both Rim101 and the repressor Nrg1. In our study, we quantitatively recorded the transcription of two native Rim101 targets, RIM8
, and assayed RNA accumulation from the native genomic loci. Both were repressed by Rim101 in WT and bro1
Δ mutant cells while being derepressed in dfg16
Δ and bro1
Δ mutant cells. Our findings indicate that a bro1
Δ mutation does not alleviate Rim101-dependent repression at all target genes.
Our observations reported here argue that association of Rim20 with endosomes has to be accompanied by an additional signal(s) mediated by alkaline pH that may change the conformation of the ESCRT complex or its interaction with Rim101 pathway components. This change mediated by a pH shift may specialize some ESCRT-endosomes as hubs for downstream signaling and open new sites to facilitate recruitment of the protease Rim13 or other factors.