Article

Intervention of Bro1 in pH-Responsive Rim20 Localization in Saccharomyces cerevisiae

Department of Microbiology, Columbia University, New York, New York 10032, USA.
Eukaryotic Cell (Impact Factor: 3.18). 02/2010; 9(4):532-8. DOI: 10.1128/EC.00027-10
Source: PubMed

ABSTRACT

Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required
for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth
conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization
of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent
protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline
conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27
but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for
RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy
exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate
Rim101, as evidenced by the expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.

Full-text preview

Available from: ncbi.nlm.nih.gov
  • Source
    • "plexes and associated proteins ( i . e . , ALIX ) . Finally , MVBs fuse with vacuoles and release ILVs to the vacuole lumen where they are degraded together with their cargoes . In alix - 1 mutants , ALIX - 1 association with ESCRT - III complexes is altered , and PHT1 ; 1 proteins are not correctly internalized into ILVs , Fisher et al . , 2007 ; Boysen et al . , 2010 ) . Mutant and structural analyses showed that whereas the second hydrophobic patch is responsible for Bro1 / ALIX binding to SNF7 / CHMP4 , the TPR pocket helps to stabilize intramolecular interactions between different parts of the Bro1 domain . Gly - 260 lays in a - helix 9 that is part of the TPR pocket . This position is relatively"
    [Show abstract] [Hide abstract]
    ABSTRACT: Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.
    Full-text · Article · Sep 2015 · The Plant Cell
  • Source
    • "plexes and associated proteins ( i . e . , ALIX ) . Finally , MVBs fuse with vacuoles and release ILVs to the vacuole lumen where they are degraded together with their cargoes . In alix - 1 mutants , ALIX - 1 association with ESCRT - III complexes is altered , and PHT1 ; 1 proteins are not correctly internalized into ILVs , Fisher et al . , 2007 ; Boysen et al . , 2010 ) . Mutant and structural analyses showed that whereas the second hydrophobic patch is responsible for Bro1 / ALIX binding to SNF7 / CHMP4 , the TPR pocket helps to stabilize intramolecular interactions between different parts of the Bro1 domain . Gly - 260 lays in a - helix 9 that is part of the TPR pocket . This position is relatively"
    [Show abstract] [Hide abstract]
    ABSTRACT: Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.
    Full-text · Article · Aug 2015 · Proceedings of the National Academy of Sciences
    • "tants , ALIX - 1 association with ESCRT - III complexes is altered , and PHT1 ; 1 proteins are not correctly internalized into ILVs , and three b - sheets that form a tetratricopeptide ( TPR ) pocket and two exposed hydrophobic patch substructures ( Odorizzi et al . , 2003 ; Kim et al . , 2005 ; Boysen and Mitchell , 2006 ; Fisher et al . , 2007 ; Boysen et al . , 2010 ) . Mutant and structural analyses showed that whereas the second hydrophobic patch is responsible for Bro1 / ALIX binding to SNF7 / CHMP4 , the TPR pocket helps to stabilize intramolecular interactions between different parts of the Bro1 domain . Gly - 260 lays in a - helix 9 that is part of the TPR pocket . This position is relatively"

    No preview · Conference Paper · Aug 2012
Show more