| dc.description.abstract | Mitochondria are critical double-membraned organelles that act as biosynthetic and bioenergetic cellular factories, with the outer membrane providing an interface with the rest of the cell. In humans, the outer mitochondrial membrane (OMM) contains ~110 different proteins which are encoded in the nuclear genome, synthesized in the cytosol and must be targeted to the membrane. OMM proteins are defined by the secondary structure of their transmembrane domains (TMDs), as two classes – beta-barrel proteins, evolutionarily derived from the outer membranes of gram-negative bacteria, and alpha-helical proteins, an evolutionarily more recent class. Beta-barrel proteins are first translocated into the mitochondrial intermembrane space (IMS) via the translocase of the outer membrane (TOM) and subsequently inserted by the sorting and assembly machinery (SAM) complex. Comparatively, alpha-helical OMM protein biogenesis is poorly understood. Alpha-helical proteins are classified as signal-anchored (a single N-terminal anchored TMD), tail-anchored (a single C-terminal anchored TMD) and polytopic (multiple TMDs), by the number and orientation of their TMDs with respect to the membrane. While the novel OMM insertase MTCH2 was discovered using a genome-wide CRISPRi screen for alpha-helical tailanchored substrates (Guna et. al, 2022), the broader biogenesis and targeting pathways for all biophysically diverse alpha-helical proteins remained unexplored. Critically, the mechanisms of cytosolic chaperoning and targeting for all alpha-helical OMM proteins were unknown. This thesis presents a large-scale investigation that systematically delineates alphahelical biogenesis pathways, from cytosolic chaperoning to membrane insertion to quality control of unassembled or mis-localized TMDs. Genome-wide CRISPRi screens in human cells for varied signal-anchored and polytopic substrates revealed novel cytosolic chaperones, targeting and quality control factors. Arrayed follow-up genetic screens against a large and biophysically more varied panel of substrates revealed that alpha-helical proteins are triaged in the cytosol by TMD number and topology, thus defining a set of ‘rules’ for biogenesis. Cell biological and in vitro biochemistry experiments further discovered a new role for the ribosome-bound chaperone NAC in regulating polytopic protein biogenesis and characterized a novel signal-anchored targeting factor TTC1 that chaperones TMDs using a conserved C-terminal hydrophobic groove. Cumulatively, this work both defines the pathways for biogenesis and quality control of alphahelical OMM proteins and identifies mechanisms by which mitochondrial protein composition and thereby function can be tuned through manipulation of mitochondrial membrane protein biogenesis machinery in diverse pathophysiological conditions. | |
