Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deficiency

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency i s an autosomal recessive neurodegenerative disorder caused by mutations in SURF1, Although SURF1 is ubiquitously expressed, its expression is lower in brain than in other highly aerobic tissues, All reported SURF1 mutations a re loss of function, predicting a truncated protein (hSurf1) product. Weste rn blot analysis with anti-hSurf1 antibodies demonstrated a specific 30 kDa protein in control fibroblasts, but no protein in LS patient cells, Steady -state levels of both nuclear- and mitochondrial-encoded COX subunits were also markedly reduced in patient cells, consistent with a failure to assemb le or maintain a normal amount of the enzyme complex. An epitope (FLAG)-tag ged hSurf1 was targeted to mitochondria in COS7 cells and a mitochondrial i mport assay showed that the hSurf1 precursor protein (35 kDa) was imported and processed to its mature form (30 kDa) in a membrane potential-dependent fashion. The protein was resistant to alkaline carbonate extraction and su sceptible to proteinase K digestion in mitoplasts, Mutant proteins In which the N-terminal transmembrane domain or central loop were deleted, or the C -terminal transmembrane domain disrupted, did not accumulate and could not rescue COX activity in patient cells. Co-expression of the N- and C-termina l transmembrane domains as independent entities also failed to rescue the e nzyme deficiency, These data demonstrate that hSurf1 is an integral inner m embrane protein with an essential role in the assembly or maintenance of th e COX complex and that insertion of both transmembrane domains in the intac t protein is necessary for function.