SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma

Abstract

Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome 11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics analysis in yeast has led to the discovery of a human tumor susceptibility gene.

Fig. 1

Sdh5 is required for respiration and interacts with Sdh1. (A) Mitochondria, mitoplasts generated by hypotonic swelling, and 1% Triton X-100–solubilized mitochondria from a strain expressing Sdh5-HA were treated with (+) or without (−) proteinase K and analyzed by immunoblotting with an untreated mitochondria control (UT). Mge1, Tim10, and Fzo1 are matrix, intermembrane space, and outer membrane proteins, respectively. (B) Soluble and membrane fractions of purified mitochondria (4) as in (A) were immunoblotted. Aco1, soluble matrix protein; Sdh1, membrane-associated matrix protein. (C) Serial dilutions of WT and sdh5Δ strains containing empty vector (EV) or a plasmid expressing Sdh5-HA were spotted on glucose or glycerol medium and grown at 30°C for 2 or 3 days, respectively. (D) Oxygen consumption in WT, sdh5Δ, and sdh1Δ strains grown to mid-log phase in raffinose media (±SD, n = 3 biological replicates). Similar results were obtained in glucose medium. (E) Tandem purification eluates (4) from a WT strain and a strain expressing Sdh5-His₆/HA were resolved with SDS-PAGE and visualized by silver staining (top panel) or immunoblot with antibodies to Sdh1 and HA (lower panels). (F) Immunoblot of purified mitochondria from WT, sdh1Δ, or sdh2Δ strains expressing Sdh5-HA. Porin, mitochondrial loading control.

Fig. 2

Sdh5 is required for SDH activity and stability. (A) SDH and malate dehydrogenase activity (4) from WT and sdh5Δ mitochondria, normalized to total protein (±SD, n = 3 biological replicates). (B) In-gel activity assay of ETC complexes after BN-PAGE of mitochondrial membranes from WT and sdh5Δ strains. V₂, complex V dimer. (C) Coomassiestained BN-PAGE of mitochondrial membranes from WT and sdh5Δ strains. (D) Immunoblot of BN-PAGE–separated complex II/SDH using an antibody to Myc to show Myc-tagged Sdh3 in WT and sdh5Δ mitochondria. (E) Immunoblot of BN-PAGE–separated WT and Sdh5-TAP mitochondria using an antibody to TAP, without and with 1% SDS pretreatment. Porin is an ~440-kD control. (F) Immunoblot of mitochondria from WT and sdh5Δ strains in which Sdh3 or Sdh4 was Myc-tagged, separated into soluble (sol) and membrane (mem) fractions (4), or unfractionated (total). Aco1, soluble matrix protein. The indicated percentage is the amount remaining in sdh5Δ mitochondria relative to WT mitochondria.

Fig. 3

Sdh5 is necessary and sufficient for Sdh1 flavination. (A) WT, sdh5Δ, and sdh1Δ mitochondria were resolved by SDS-PAGE and imaged (4) for covalent FAD (top panel) or immunoblotted (lower panels). (B) Fluorescence gel image (top panel) and immunoblot (lower panels) as in (A), with whole-cell extract from WT or flx1Δ sdh5Δ strains containing EV, CEN plasmid SDH5 (flx1Δ: ~1 copy per cell), or 2μ plasmid SDH5 (O/E: ~10 copies per cell). The bar graph shows normalized FAD fluorescence (±SD, n = 3 biological replicates) (bottom panel). (C) His-tagged yeast Sdh1 was expressed alone or with Sdh5 or Sdh2 in E. coli, purified, and analyzed for FAD fluorescence as in (A) and by Coomassie blue staining.

Fig. 4

A human SDH5 loss-of-function mutation in PGL2. (A) The heterozygous c.232G>A mutation segregates with disease in the PGL2 lineage (17). Black symbols, affected persons; white symbols, unaffected persons; +, heterozygous mutation; NT, not tested. Diamonds with the number 4 represent four unaffected individuals. Individuals who are not affected because they carry the mutation on their maternal chromosome 11 are marked by an m. One healthy maternal mutation carrier and five non-affected paternal mutation carriers are not shown in the pedigree for privacy reasons. (B) Fluorescence gel image (top panel) and immunoblotting (lower panels) of samples from human tumors, cell lines, and mouse tissues. Lanes 1 and 2, sporadic PGL tumors; lanes 3 to 5, PGL2 tumors (hSDH5 G78R); lanes 6 and 7, HEK293 and HepG2 human cell lines; lanes 8 and 9, normal mouse skeletal muscle (skM) and liver. (C) Lysate from HEK293 cells containing EV or expressing WT or G78R hSDH5-Myc were immunoprecipitated with agarose beads conjugated with antibody to Myc. Lysate, eluate, and unbound fraction were FAD-imaged (top panel) and immunoblotted (lower three panels). (D) Serial dilutions of WT and sdh5Δ strains containing EV or plasmids expressing yeast Sdh5-Myc, WT human SDH5-Myc, or G78R hSDH5-Myc were spotted on glucose or glycerol medium and grown at 30°C for 2 or 3 days, respectively. (E) Fluorescence gel image (top panel) and immunoblotting of whole-cell extract from the five strains in (D) (lower panels). PGK, loading control. FAD fluorescence was normalized to Sdh1 protein level and expressed as a percentage relative to WT (Flavo%, ±SD, n = 3 biological replicates).