FUNCTIONAL-ANALYSIS OF A RECENTLY ORIGINATING, ATYPICAL PRESEQUENCE -MITOCHONDRIAL IMPORT AND PROCESSING OF GUS FUSION PROTEINS IN TRANSGENIC TOBACCO AND YEAST

A gene family of at least five members encodes the tobacco mitochondri al Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichin coli beta-glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS pro teins were localized by cell fractionation. The aminoterminal 75 and 7 1 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-term inal sequence of RISP4, which contains an atypical mitochondrial prese quence, can translocate the GUS protein specifically into tobacco mito chondria with apparently low efficiency. Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the t obacco RISP3 and RISP4 presequences can direct import and processing o f a GUS fusion protein in yeast mitochondria. Plant presequences, howe ver, direct mitochondrial import in yeast less efficiently than the ye ast presequence, indicating subtle differences between the plant and y east mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an a mphipathic secondary structure; however, these structural properties m ay improve the efficiency of mitochondrial import.