Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking

Abstract

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

How will I recognize you? Insights into endocytic cargo recognition in plants

“…OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 have been shown to tune down the internalization of the auxin efflux carrier PIN2. Their DUB activity is stimulated by the presence of anionic lipids, which likely causes a conformational change to increase the accessibility of ubiquitin to the catalytic site [67]. The above result indicates that ubiquitination is counteracted at the PM.…”

Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation

“…The NOS terminator was amplified from the vector pGWB411 with primers MN447/MN448 and cloned into pJET1.2 to generate pMN174. The constructs pMN173, pMN174, and pMN175 together with the vector pBB02 71 were used for Golden Gate assembly with either pMN199 or pMN200.…”

“…Immunoblotting was conducted using the following primary antibodies: anti-ALIX 14 , anti-H + -ATPase (5000× diluted, Agrisera, AS07260), and anti-FLAG (M2) (1000×diluted, Merck, F1804), anti-GBD (3000× diluted, Santa Cruz, sc-510), anti-GFP (1000× diluted, 3H9, Chromotek, 3H9-100), anti-RFP (1000× diluted, 6G6, Chromotek, 6G6-150), anti-GST (1000× diluted, Eurogentec) 71 , anti-His (1000× diluted, Thermo Fisher, P-21315), anti-HA(3F10) (1000× diluted, Roche, 11867423001), anti-MBP (10000× diluted, NEB, E8032S), anti-UGPase (3000× diluted, Agrisera, AS05086), anti-NBR1 (2000× diluted, Agrisera, AS142805A), anti-H3 (5000× diluted, Agrisera, AS10710), anti-ACTIN (50× diluted, JLA20, Merck, MABT219), anti-CDC2 (5000× diluted, Santa Cruz, sc-166885). As secondary antibodies, anti-rat-HRP (80000× diluted, Merck, A9037), anti-mouse-HRP (80000×diluted, Merck, A9044), anti-rabbit-HRP (80000×diluted, Merck, A0545), anti-rabbit-Alkaline Phosphatase (AP) (30000× diluted, Merck, A3812), and an anti-rat-AP (30000× diluted, Merck, A6066) were used and to detect protein bands in an Amersham TM Imager 600 (Cytiva).…”

Identification of promoter targets by Aureochrome 1a in the diatom Phaeodactylum tricornutum

“…His6- and GST-tagged proteins and GST protein were detected using primary and secondary antibodies listed below with the following dilution. Primary antibodies: mouse anti-Penta-His tag monoclonal antibody (1:1000) (P-21315, Thermo Fisher Scientific); rabbit anti-GST antibody (1:1,000) (Eurogentec) is a custom-made antibody as described in Vogel et al (2022) . Secondary antibodies: anti-mouse–horseradish peroxidase (HRP) (1:80 000) (A9044, Sigma Aldrich); anti-rabbit–HRP (1:80 000) (A0545, Sigma Aldrich).…”