Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast

Surf1p is a protein involved in the assembly of mitochondrial respiratory c hain complexes, However its exact role in this process remains to be elucid ated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidas e (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients, Asse mbly of COX was analysed in a shy1 null mutant strain by two-dimensional po lyacrylamide gel electrophoresis (2D-PAGE). Steady-state levels of the enzy me were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount o f holo-COX in the SHY1-disruptant strain suggests that Shy1p mag either fac ilitate assembly of the enzyme, or increase its stability, However, our obs ervations, based on 2D-PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Delta shy1 strain, COX enzyme assembled in the absence of Shy1p appears to be st ructurally and enzymically normal, The in vitro labelling studies additiona lly indicate that mitochondrial translation is significantly increased in t he shy1 null mutant strain, possibly reflecting a compensatory mechanism fo r reduced respiratory capacity. Protein interactions of both Shy1p and Surf 1p are implied by their appearance in a high molecular weight complex of ab out 250 kDa, as shown by 2D-PAGE. (C) 2001 Federation of European Microbiol ogical Societies. Published by Elsevier Science E,V, AII rights reserved.