Expression of the biotin-containing MCCα subunit in fibroblasts. Proteins in mitochondrial enriched fractions from cultured fibroblasts were separated by SDS-PAGE, and the biotin-containing subunits of MCC, PCC, and PC were detected with an avidin alkaline phosphatase conjugate. Most, but not all, CG2-probands lack the MCCα subunit, while it is detectable in all CG1 probands.

Coomassie-stained SDS-PAGE gel of biotin-containing proteins purified from a mouse kidney extract with streptavidin Dynabeads. We excised the proteins from the gel, digested with trypsin, and analyzed the tryptic peptides using MALDI-TOF and liquid chromatography coupled to ESI-MS/MS. We analyzed all the indicated bands including those that we identified as MCCα and β.

The MCC-catalyzed reaction and its position in the leucine catabolic pathway. The dashed arrow indicates the metabolites that accumulate due to deficiency of MCC.

Sequence alignment of human MCCα and MCCβ with orthologs from mouse and A. thaliana. Amino acids identical to the human sequence are highlighted. Missense mutations identified in MCC-deficient patients are indicated above with the substituted amino acid. Potential cleavage sites for the NH₂-terminal mitochondrial leader sequences are indicated by vertical arrowheads. (a) Sequence alignment of human, mouse, and A. thaliana MCCα. The predicted ATP-binding site in the NH₂-terminal biotin carboxylation domain and the predicted COOH-terminal biotin carboxyl carrier domain are indicated by solid and dashed over-lines, respectively. The arrow indicates the lysine residue that links covalently to biotin (biocytin). The residues marked with an asterisk within the biotin carboxylation domain are thought to play an important role in catalysis (36, 37). (b) Sequence alignment of human and A. thaliana MCCβ. The putative 3-methylcrotonyl-CoA binding domain is indicated by a solid over-line.

MCCA and B missense mutations that alter splicing. (a) MCCA D532H. The 1594G→C transversion of the last bp of exon 13 results in the missense mutation D532H. The 3′ base of an exon also contributes to donor splice site recognition and, as shown in the lower panel, RT-PCR of MCCA cDNA in this patient with primers corresponding to the 5′ and 3′ UTR resulted in a product smaller than in wild-type. Sequence analysis of this product showed that exon 13 (217 bp) is skipped, which shifts the reading frame. Thus, the deleterious consequences of this missense mutation appear to be entirely due to the splicing defect. (b) MCCB I437V. As shown in the upper panel, the 1309A→G transition in exon 14 results in the replacement of isoleucine by valine, a conservative change. However, the mutation also activates a cryptic splice donor. Use of this new donor splice site deletes the last 64 bp of exon 14 from the mature transcript. As shown in the lower panel, direct sequencing of the RT-PCR product shows that virtually all the transcript present uses the new, more 5′ splice donor. The second allele of this compound heterozygous patient does not produce detectable RNA. wt, wild-type; mut, mutant.