An ubiquitin-dependent balance between mitofusin turnover and fatty acids desaturation regulates mitochondrial fusion

Abstract

Mitochondrial integrity relies on homotypic fusion between adjacent outer membranes, which is mediated by large GTPases called mitofusins. The regulation of this process remains nonetheless elusive. Here, we report a crosstalk between the ubiquitin protease Ubp2 and the ubiquitin ligases Mdm30 and Rsp5 that modulates mitochondrial fusion. Ubp2 is an antagonist of Rsp5, which promotes synthesis of the fatty acids desaturase Ole1. We show that Ubp2 also counteracts Mdm30-mediated turnover of the yeast mitofusin Fzo1 and that Mdm30 targets Ubp2 for degradation thereby inducing Rsp5-mediated desaturation of fatty acids. Exogenous desaturated fatty acids inhibit Ubp2 degradation resulting in higher levels of Fzo1 and maintenance of efficient mitochondrial fusion. Our results demonstrate that the Mdm30-Ubp2-Rsp5 crosstalk regulates mitochondrial fusion by coordinating an intricate balance between Fzo1 turnover and the status of fatty acids saturation. This pathway may link outer membrane fusion to lipids homeostasis.

A membrane-inserted structural model of the yeast mitofusin Fzo1

“…Both the Fzo1 D335K single mutant and, most surprisingly, the D335K-K464D double mutant (charge swap) displayed levels comparable to the wild-type protein. This observation led to compare the ubiquitylation status of single ( K464D ) or double swap ( D335K-K464D ) Fzo1-13Myc mutants with that of K398R that is established to alter ubiquitylation of the mitofusin 15 , 35 . As expected, detection of the Mdm30-dependent ubiquitylation doublet was strongly impaired in K464D and K398R mutants (Fig.…”

“…Alternatively, this restored proximity may contribute to maintain a correct fold of Fzo1 that would favor ubiquitylation on K398. The latter possibility is more likely as K398 is the main target for Mdm30-dependent ubiquitylation of Fzo1 35 . In aggregate, these results provide experimental confirmation of a physical interaction between the two residues located in the N- and C-terminal halves of Fzo1 and contribute to validating the predicted proximity in our model.…”

Regulation of lipid saturation without sensing membrane fluidity

“…The assays and tools established here provide handles to better understand the structural and dynamic features that render a protein a good substrate of the E3 ubiquitin ligase Rsp5. Identifying the molecular rules of substrate selection is a major open question, because Rsp5 has been implicated in most diverse aspects of cellular physiology including endocytosis 58 , mitochondrial fusion 62 , and the turnover of heat-damaged proteins in the cytosol 63 . Our in vitro system using a membrane-reconstituted, conditional substrate of Rsp5 provides a unique opportunity to better understand (i) the contribution of trans-autoubiquitylation of Rsp5, (ii) the relevance of structural malleability in Rsp5 substrates, and (iii) the role of deubiquitylating enzymes in defining the selectivity and sensitivity of the Rsp5-mediated ubiquitylation.…”

Regulation of lipid saturation without sensing membrane fluidity

“…The assays and tools established here, provide new handles to better understand the structural and dynamic features that render a protein a good substrate of the E3 ubiquitin ligase Rsp5. Identifying the molecular rules of substrate selection is a major open question, because Rsp5 has been implicated in most diverse aspects of cellular physiology including endocytosis 52 , mitochondrial fusion 58 , and the turnover of heat-damaged proteins in the cytosol 56 . Our in vitro system using a membrane-reconstituted, conditional substrate of Rsp5 provides a unique opportunity to better understand i) the contribution of trans -autoubiquitylation of Rsp5, ii) the relevance of structural malleability in Rsp5 substrates, and iii) the role of deubiquitylating enzymes in defining the selectivity and sensitivity of the Rsp5-mediated ubiquitylation.…”