Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression (original) (raw)
Figure 6
UQCC2 interacts with mitochondrial protein UQCC1.
(A) SDS-PAGE analysis of HEK293 cellular fractions shows that UQCC1 is enriched in the mitochondrial fraction, similar to the mitochondrial protein TOM20. A cytosolic marker creatine kinase B-type (CK-B) was used. TC: Total Cell, Cyt: Cytoplasmic fraction, Mit: Mitochondrial fraction. (B) Proteinase K protection assay performed using mitochondria with digitonin-permeabilized outer membranes shows localization of UQCC1 within the mitochondrial inner membrane. UQCC1, unlike outer membrane localized TOM20 and the inter-membrane localized part of OXA1L, is protected from proteolysis and degraded only after the inner membrane is dissolved with Triton X-100. Western blot analysis of single step affinity purified (C) UQCC2- and (D) UQCC1-TAP from doxycycline-induced HEK293 cells shows that UQCC1 co-purifies with UQCC2-TAP and UQCC2 co-purifies with UQCC1-TAP. Additional probing of the membranes for the complex III structural subunits UQCRC1, UQCRC2, UQCRFS1 and mitochondrial ribosomal subunits MRPS22 and MRPL12 did not reveal co-elution of these proteins. Asterisks with these subunits, including the one with UQCRFS1, correspond to bands at different heights that result from previous incubations. Complex II subunit SDHA was used to rule out non-specific protein binding. Non-induced cells were used as control. Antibodies used are indicated at the left. Arrowheads indicate endogenous UQCC1 and UQCC2.