Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3.
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.
Plant Systems Biology, Technische Universität München, 85354 Freising, Germany;
Kamila
K
Kalinowska
Plant Systems Biology, Technische Universität München, 85354 Freising, Germany;
Marie-Kristin
MK
Nagel
Departments of Botany and Genetics, Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706;
Kaija
K
Goodman
Centro Nacional de Biotecnología-CSIC, 28049 Madrid, Spain.
Laura
L
Cuyas
Plant Systems Biology, Technische Universität München, 85354 Freising, Germany;
Franziska
F
Anzenberger
Plant Systems Biology, Technische Universität München, 85354 Freising, Germany;
Angela
A
Alkofer
Centro Nacional de Biotecnología-CSIC, 28049 Madrid, Spain.
Javier
J
Paz-Ares
Plant Systems Biology, Technische Universität München, 85354 Freising, Germany;
Pascal
P
Braun
Centro Nacional de Biotecnología-CSIC, 28049 Madrid, Spain.
Vicente
V
Rubio
Departments of Botany and Genetics, Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706;
Marisa S
MS
Otegui
Plant Systems Biology, Technische Universität München, 85354 Freising, Germany; erika.isono@wzw.tum.de.
Erika
E
Isono
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D011485:Protein Binding; D036226:Seedlings / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure
C000606499:ALIX protein, Arabidopsis; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008164:Luminescent Proteins; D025801:Ubiquitin; D064570:Ubiquitin-Specific Proteases; C586776:AMSH3 protein, Arabidopsis
Arabidopsis; ESCRT-III; intracellular trafficking; ubiquitin