Fig. 2.

LIX1 mitochondrial localization is controlled by S-palmitoylation at Cys84.

(A) Sequence alignment of human LIX1 and its orthologs. Asterisks indicate the position of the cysteine residues corresponding to putative S-palmitoylation sites. (B) LIX1 S-palmitoylation analysis with the Acyl RAC method. Membranes were incubated with anti-HA tag antibodies to detect LIX1. IF, input fraction; cUF, cleaved unbound fraction; cBF, cleaved bound fraction; pUF, preserved unbound fraction; pBF, preserved bound fraction. (C) Representative Western blot analysis of total extracts and isolated mitochondria from the same amount of total extract from HeLa cells that express WT HA-LIX1 (LIX1 WT), single LIX1 mutants (LIX1 C83S, LIX1 C84S) or the double mutant (LIX1 C83S/C84S) with antibodies against the HA tag and TFAM (matrix protein). (D) Quantification of the western blots showing the percentage of LIX1 (normalized to TFAM level) in mitochondria relative to LIX1 level in the total extract (normalized to TFAM level). (E) 3D structure of the LIX1 protein showing that the cysteine residue 84 is accessible for modification.