The ER-associated E3 ligase RMA1 is a novel regulator of the autophagy machinery

Autophagy is a fundamental cellular pathway that mediates the degradation and recycling of cytoplasmic components. By ensuring protein and organelle quality control, it plays a central role in maintaining cellular homeostasis, adapting to metabolic demands, and protecting against environmental stresses. This tightly regulated process is essential not only for cell survival under nutrient limitation but also for development, immunity, and disease prevention. Notably, SH3P2 participates in the early stage of autophagy, particularly during phagophore formation. To date, the only known regulator of SH3P2 is a bacterial E3 ligase that triggers its proteasomal degradation during infection. Identifying in planta regulators of SH3P2 is therefore essential to understand membrane-dependent regulation of cellular homeostasis. Analysis of the SH3P2 interactome under proteasomal inhibition revealed a concomitant enrichment of plasma membrane proteins and depletion of secretory proteins. We also identified a potential novel regulator, the RING E3 ligase RMA1, belonging to a clade whose animal members (RNF5/RNF185) have been characterized as autophagy regulators. Subcellular localization studies showed that RMA1 is a dynamic protein, defined by endoplasmic reticulum localization and vesicle-like structures. Domain characterization revealed that its E3 activity and membrane anchoring regulate its dynamics and stability, which correlate with its ability to associate with SH3P2. Consistently, RMA1 physically interacts with and ubiquitinates SH3P2, as well as other key autophagy components. Our results further suggest that RMA1 regulation is linked to mitochondrial stress responses, highlighting a potential role in mitophagy. Taken together, we identify RMA1 as a novel regulator of the autophagic machinery.