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Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact role of Miro in these functions is unclear but involves binding to a variety of client proteins. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro interactors—namely, CENPF, Trak, and MYO19—all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the first calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including MTFR1/2/1L, the lipid transporters Mdm34 and VPS13D, and the ubiquitin E3-ligase Parkin. Given the shared binding mechanism of these functionally diverse clients and its conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health. Synopsis: Miro GTPases control many aspects of mitochondrial function by recruiting diverse proteins. Here, the Miro proteins are shown to interact with their effectors via a conserved hydrophobic pocket, enabling their function as general mitochondrial adaptors. Miro proteins contain a hydrophobic cavity (ELF pocket) in their first ELM1 domain that is required for client binding. All known Miro clients use a highly conserved leucine or phenylalanine residue to bind the Miro ELF pocket. Auxiliary binding features, such as salt bridges and trans β-strands, modulate Miro interaction with client proteins. Binding configuration between functionally diverse Miro clients is conserved in eukaryotes. Miro GTPases use a highly conserved hydrophobic pocket to interact with functionally diverse effector proteins. [ABSTRACT FROM AUTHOR]

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non‐vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1—an uncharacterized protein harboring a Chorein‐N lipid transport motif—for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation. Synopsis: How membrane composition and lipid transport are controlled in eukaryotic cells remains poorly understood. Here, a comprehensive screen on rerouted phospholipid biosynthesis in yeast uncovers principles of lipid distribution and plasticity. Engineering chimeric enyzmes targeted to specific organelles allows for generation of 24 yeast strains with confined phosphatidylethanolamine and phosphatidylcholine biosynthesis.Yeast cells can tolerate topological rewiring of lipid biosynthesis.A mutagenesis screen identifies factors required for survival upon rewired lipid biosynthesis.Similar transposon insertion patterns across rewired conditions reveal clusters of functionally related genes.The lipid transport protein Csf1 is required for lipidomic adaptation to cold stress. [ABSTRACT FROM AUTHOR]