Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPR^mt in muscle.

The mitochondrial unfolded protein response (UPR^mt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Because of the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mitonuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPR^mt. Thus, we sought to perturb this genomic coordination by using a systemic antisense oligonucleotide (in vivo morpholino) targeted to translocase of the inner membrane channel subunit 23 (Tim23), the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate reactive oxygen species emission under maximal respiration conditions. This import defect activated the C/EBP homologous protein (CHOP) branch of the UPR^mt, as evident from increases in caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and chaperonin 10 (cpn10) but not the activating transcription factor 5 (ATF5) arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via emission, to modify nuclear gene expression and ultimately improve folding capacity.

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