Abstract
CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.
Keywords:
ANT, adenine nucleotide translocator; Baf, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CHX, cycloheximide; FB1, FAD/NAD (P)-binding domain 1; FB2, FAD/NAD (P)-binding domain 2; IM, inner membrane; IMS, inter-membrane space; LC3; MPP+, 1-methyl-4-phenylpyridinium; MTS, mitochondrial targeting sequence; Mat, matrix; OM, outer membrane; PARK2/parkin; PB1, Phox and Bem 1 domain; PD, Parkinson disease; PK, proteinase K; RD, FAD-linked reductase domain; SQSTM1/p62; choline dehydrogenase; mitophagy.