Analysis of the dual function of the ESCRT-III protein Snf7 in endocytic trafficking and in gene expression.
ESCRT (endosomal sorting complex required for transport)-III mediates the budding and scission of intralumenal vesicles into multivesicular endosomes in yeast. For the main ESCRT-III subunit Snf7, an additional role in activation of the transcription factor Rim101 (the 'Rim pathway') is now also firmly established. In the present study, we investigate how these two Snf7 functions are related to each other. By generating SNF7 mutations that severely affect endocytic trafficking, but leave the Rim pathway function intact, we show that the two functions of SNF7 can be separated genetically. We analysed in detail how the SNF7 mutations affect the interaction of Snf7 with its various binding partners. Although the interactions with proteins Rim13 and Rim20, necessary for the Rim-pathway-related functions, were not altered by the mutations, there was a strong effect on interactions with components of the ESCRT pathway. The interactions, as measured by co-immunoprecipitation, with the ESCRT-III subunits Vps20 and Vps24 were strongly increased by the mutations, whereas the interactions with proteins Vps4 and Bro1, acting downstream of ESCRT-III, were reduced. As Vps4 is required for disassembly of ESCRT-III these results suggest that ESCRT-III is more stable in our SNF7 mutants. In line with this notion, a higher fraction of mutant Snf7 protein was detected at the membrane. Upon a shift to alkaline pH, a stronger binding signal for virtually all interaction partners, except Vps4, was observed. This indicates that the ESCRT network at the endosomal membrane is more extensive under these conditions.
Weiss
Peter
P
Institut für Lebensmittelwissenschaft und Biotechnologie, Fg. Gärungstechnologie, Universität Hohenheim, D-70599 Stuttgart, Germany.
Huppert
Stefanie
S
Kölling
Ralf
R
D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology
C480237:Bro1 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; C510979:Rim20 protein, S cerevisiae; C083890:SNF7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins