The ability to adapt to diverse environmental conditions is critical for the survival of free-living microorganisms. The response to an environmental change is typically orchestrated by signal transduction pathways. Functional outputs may include both transcriptional and posttranscriptional changes that alter microbe physiology, establishing a state that improves growth under the altered environmental conditions (1
Our focus is the adaptation of fungi to changes in environmental pH. A signal transduction pathway, the Rim101/PacC pathway, is broadly conserved among fungi (6
). It mediates diverse cellular behaviors that include salt tolerance and sporulation in Saccharomyces cerevisiae
, secondary metabolite production in A. nidulans
, and pathogenicity in Candida albicans
, Aspergillus fumigatus
, and Cryptococcus neoformans
). Many environmental response pathways comprise mitogen-activated protein kinase modules, but the Rim101/PacC pathway uses a different signaling mechanism. The key pathway output is the activation of a transcription factor, called Rim101 or PacC, by C-terminal proteolytic cleavage. The cleavage reaction is tied to endocytic vesicle metabolism. Recent observations indicate that other signaling pathways are also intimately connected to vesicle metabolism (19
). The Rim101/PacC pathway may serve as a model for this emerging regulatory paradigm.
The Rim101/PacC signaling pathway may be viewed as two modules (18
). The upstream module is the “sensing complex,” which includes transmembrane proteins Dfg16, Rim21, and Rim9, along with the β-arrestin homolog Rim8. Signal recognition results in activation of Rim8 through phosphorylation and ubiquitination (11
). Activation of Rim8 stimulates function of the downstream module, which is the “proteolytic complex.” This complex includes scaffold protein Rim20, protease homolog Rim13, endosomal ESCRT, and nascent transcription factor Rim101. Rim20 binds to the Rim101 C-terminal region and promotes its cleavage by Rim13. The N-terminal region of Rim101 includes three zinc fingers. Rim101 functions as a transcriptional repressor in S. cerevisiae
), while many Rim101/PacC proteins in other fungi are transcriptional activators (23
The functional connection of the proteolytic complex to endocytic vesicles is indicated by four lines of evidence. First, Rim101 cleavage depends upon several endocytic vesicle proteins, including Snf7, that are subunits of the ESCRT complex (10
). Second, Rim101 cleavage becomes independent of the upstream sensing complex in strains that hyperaccumulate ESCRT complexes, such as vps4Δ
). Third, two-hybrid studies indicate that Rim20 and Rim13 can each interact with Snf7 (14
). Fourth, Rim20-GFP is localized to punctate structures when the Rim101 pathway is activated by alkaline conditions (3
), and these structures have properties of endocytic vesicles: they are stained by the lipophilic dye FM4-64, they are labeled by Snf7-RFP, and they hyperaccumulate in a vps4Δ
mutant strain. In addition, punctate localization of Rim20-GFP depends upon Snf7 and several other ESCRT subunits. Therefore, activation of the Rim101 proteolytic complex is thought to occur through association of Rim20 with the ESCRT complex on endocytic vesicle surfaces.
Rim13 was first thought to associate with the Rim20-ESCRT complex because it interacts with Snf7 in two-hybrid assays (14
). Strong evidence in support of this association comes from a recent study of the A. fumigatus
Rim13 ortholog, PalB (25
). PalB has an N-terminal MIT domain, a feature found in ESCRT-interacting proteins (13
). The study showed that PalB is membrane associated and that it interacts directly with ESCRT subunit Vps24. Both of these interactions depend upon the PalB MIT domain. In addition, PalB function is partially impaired by an MIT domain deletion, and full function of PalB lacking its MIT domain was recovered when it was expressed as a protein fusion to Vps24. This fusion restores membrane association as well, likely by restoring association with the ESCRT complex. This study clearly established a key role for the PalB MIT domain in Rim101/PacC pathway signaling and in PalB-ESCRT association.
Rim13 is distinct from PalB in lacking an MIT domain. In addition, prior two-hybrid and coimmunoprecipitation studies indicate that Rim13 interacts with Snf7 rather than Vps24 (14
). Here, we have used a combination of fluorescence microscopy and mutational analysis to define the localization of Rim13 and its functional determinants. Our results support the model that Rim13 is associated with the ESCRT complex and provide insight into the genetic and environmental regulation of Rim13-ESCRT association.