Ribosome-Associated GTPase Drg1 Links Cytosolic Translation to Mitochondrial Homeostasis

Abstract

Coordination of the functionalities among key organelles is essential for maintaining the complexity and adaptability of the eukaryotic cell. Functionally versatile proteins, such as GTPases, often assume important roles in this process. A ribosome-binding GTPase called Developmentally-regulated GTP-binding (Drg) protein is a conserved family of GTPases that promotes protein synthesis and regulates cytoskeletal dynamics, suggesting their importance for cell growth and proliferation. Mitochondrial proteostasis requires coordination between cytosolic translation and the targeting, import, and assembly of nuclear-encoded mitochondrial proteins. However, the ribosome-associated factors that support this coordination remain incompletely understood. Here, we identify the conserved GTPase Drg1, one of the two Drg paralogues in eukaryotic cells, as a translation factor required for mitochondrial homeostasis and cellular fitness. Human Drg1 and its cofactor Dfrp1 are associated with translating ribosomes and co-sedimented with polysomes. Deletion of Drg1 in HEK293T cells caused altered mitochondrial architecture, reduced mitochondrial membrane potential and ATP production, and impaired expression of a mitochondrially targeted reporter. Re-expression of wild-type Drg1 restored ATP production and reporter expression, whereas a GTPase-deficient Drg1 mutant failed to provide comparable rescue in ATP production, demonstrating that Drg1 catalytic activity is required for its cellular function. Mitochondria-directed APEX2 proximity proteomics further connected Drg1-dependent translation to proteins involved in mitochondrial protein import, respiratory-chain function, and mitochondrial protein synthesis. Reporter analyses indicated that Drg1 loss preferentially compromises productive translation toward the 3' region of the coding sequence. Together, these findings establish Drg1 as a ribosome-associated translation factor that supports the synthesis and homeostasis of proteins required for mitochondrial function. At the molecular level, loss of cellular homeostasis that is caused by compromised translation of cytoplasmically synthesized proteins functionally related to mitochondria underpins the observed phenomenon. Our results reveal a previously underappreciated connection between the cytosolic translation machinery and mitochondrial maintenance and provide a framework for understanding how defects in a conserved translational GTPase impair cellular energy metabolism and physiology.