Kamila Kalinowska, Marie-Kristin Nagel, Kaija Goodman, Laura Cuyas, Franziska Anzenberger, Angela Alkofer, Javier Paz-Ares, Pascal Braun, Vicente Rubio, Marisa S. Otegui, and Erika Isono

Significance

The regulation of protein abundance of receptors and transporters at the plasma membrane is important for proper signaling in many biological pathways. The removal of plasma membrane proteins can occur via the endocytic protein degradation pathway, in which posttranslational modification by ubiquitin plays a key role. The activity of ubiquitinating and deubiquitinating enzymes can determine the ubiquitination status of a given target protein, and it has been shown that both classes of enzymes have important physiological roles. However, how these enzymes themselves are regulated at the molecular level has not yet been completely understood. In this study, we report a possible mechanism by which the deubiquitinating enzyme AMSH3 is regulated by its interacting protein, apoptosis-linked gene-2 interacting protein X (ALIX), in Arabidopsis.

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.

See: http://www.pnas.org/content/112/40/E5543.abstract.html?etoc

PNAS October 6, 2015;   vol. 112 no. 40 E5543–E5551   

Fig. S2.

Complementation of alix-2 with ALIXpro:GFP-ALIX. (A) ALIXpro:GFP-ALIX complements seedling lethality of alix-2. Wild-type (WT), heterozygous alix-2 (hetero), and homozygous alix-2 plants expressing GFP-ALIX are shown. (Scale bar, 5 cm.) (B) Immunoblot analysis of total extracts from wild-type (WT), alix-2 with and without the ALIXpro:GFP-ALIX construct with an anti-ALIX antibody. Positions of endogenous ALIX and GFP-ALIX are indicated on the right of the panel. CDC2 was used as loading control.