Biosynthesis and processing of the large and small subunits of succinate dehydrogenase in cultured mammalian cells
George H. D. CLARKSON,* Tsoo E. KINGt and J. Gordon LINDSAY*
*Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K., and tDepartment of Chemistry, State University of New York at Albany,Albany, NY 12222, U.S.A.
Monospecific polyclonal antisera have been raised to purified bovine heart succinate dehydrogenase and to the individual large and small subunits of this enzyme. These antisera exhibit cross-reactivity with the corresponding polypeptides in rat liver (BRL), pig kidney (PK-15) and bovine kidney (NBL-1) cell lines, and were employed to investigate some of the eventsinvolved in the biogenesis of succinate dehydrogenase in the PK-15 cell line. Newly-synthesized forms of the large and small subunits of succinate dehydrogenase were detected in cultured PK-1 5 and BRL cells labelled with [35S]methionine in the presence of uncouplers of oxidative phosphorylation. In PK-15 cells, the precursor forms of the large and small subunits exhibit Mr values approx. 1000-2000 and4000-5000 greater than those of the corresponding mature forms. When the uncoupler is removed in pulse-chase experiments, complete conversion of the precursors to the mature forms occurs within 45 min. Studies on the kinetics of processing and stability of the large subunit precursor revealed that reversal of precursor accumulation is rapid, with processing occurring with a half-time of 5-7.5min, and that the accumulated precursor exhibits long-term stability when PK-15 cells are maintained in the presence of 2,4-dinitrophenol.
INTRODUCTION Succinate dehydrogenase (SDH) occupies a unique position in energy-yielding metabolism, being a component of both the tricarboxylic acid cycle and the electron transport chain. Although the purified enzyme is capable of reducing artificial electronacceptors such as ferricyanide, it requires to be associated with one or two additional polypeptide components of complex II to interact with its physiological acceptor ubiquinone (Capaldi et al., 1977; Ackrell et al., 1980; King, 1982). The enzyme from bovine heart mitochondria has been isolated in water-soluble form and consists of two subunits with Mr values of 70000 and 27000 (Davis & Hatefi,1971). The Mr 70000 polypeptide contains one molecule of covalently-attached FAD (Walker & Singer, 1970). Biophysical methods have revealed the existence of Fe-S clusters of the [2Fe-2S], [3Fe-xS] and [4Fe-4S] types, although definitive evidence concerning their subunit location has yet to emerge [for a review, see Singer & Johnson (1985)]. The enzyme is tightly associated with the mitochondrialinner membrane and exhibits an asymmetrical distribution within the lipid bilayer; ferricyanide-binding, immunological and chemical-labelling studies have indicated that SDH is located both physically and functionally on the matrix side of the inner membrane (Klingenberg & Buchholz, 1970; Merli et al., 1979; Girdlestone et al., 1981). The process whereby nuclear-coded mitochondrial polypeptides areimported into mitochondria from their cytoplasmic site of synthesis has been widely studied in recent years. In the majority of cases, these polypeptides are synthesized as higher-Mr precursor forms which possess a cleavable N-terminal signal sequence (Reid, 1985). Uptake of precursors which are destined for
insertion into or translocation across the mitochondrial inner membrane requires anelectrochemical potential gradient across the inner membrane (Schleyer et al., 1982; Gasser et al., 1982) and is accompanied by processing of the precursor to the mature polypeptide. The final step in the biogenesis of an imported mitochondrial polypeptide is the acquisition of a functional conformation in the correct submitochondrial location. The biosynthesis of SDH is of special interest since...