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The cleavable pre‐sequences of imported chloroplast and mitochondrial proteins have several features in common. This structural similarity prompted us to test whether a chloroplast pre‐sequence (‘transit peptide’) can also be decoded by the mitochondrial import machinery. In the green alga, Chlamydomonas reinhardtii, the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) (a chloroplast protein) is nuclear‐encoded and synthesized in the cytosol with a transient pre‐sequence of 45 residues. The 31 amino‐terminal residues of this chloroplast pre‐sequence were fused to mouse dihydrofolate reductase (a cytosolic protein) and to yeast cytochrome oxidase subunit IV (an imported mitochondrial protein) from which the authentic pre‐sequence had been removed. The chloroplast pre‐sequence transported both attached proteins into the yeast mitochondrial matrix or inner membrane, although it functioned less efficiently than an authentic mitochondrial pre‐sequence. We conclude that mitochondrial and chloroplast pre‐sequences perform their function by a similar mechanism.

Cytochrome b6from spinach chloroplasts (either within the purified cytochrome b6fcomplex, or in its isolated form) exhibits two spectral species, which correspond to two midpoint potentials. This can be demonstrated by low temperature difference spectroscopy at fixed redox potentials. The high potential form of cytochrome b6has a split α-peak at 557.5 and 561.5 nm, the low potential form has a symmetrical α-peak at 560.5 nm. Similar results were obtained with cytochrome b6in the isolated cytochrome b6fcomplex from the cyanobacterium Anabaena variabilis.

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines Ψ38and Ψ39in tRNA whereas the other activities, specific for Ψ formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. colias well as a protein fusion with protein A in yeast display the enzymatic activity only toward Ψ38and Ψ39formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of theDEG1gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 °C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of Ψ38- and Ψ39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truAfrom E. coli(initially called hisTor PSU-I gene product).

The cleavable prepiece of the precursor to yeast cytochrome coxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.

When the cytochrome b6/ fcomplex from chloroplasts is incorporated into liposomes, reduction of external plastocyanin by plastoquinol is stimulated under uncoupling conditions. An extra H +/e −is ejected from the vesicles during the reaction, in addition to the scalar proton liberated from plastoquinol. This is stimulated by valinomycin/K +and abolished under uncoupling conditions. Furthermore, the formation of a membrane potential during the reaction, negative inside the vesicles, is observed with the help of carbocyanine dyes. We conclude that the cytochrome b6/ fcomplex, like the cytochrome bc1from mitochondria, functions as an electrogenic proton translocator.

A photoactive P840 protein complex and a fraction enriched in cytochrome bfrom chromatophores of Chlorobium limicolaf. thiosulfatophilumare described. The former is identical in pigment composition to the ‘core complex’ of Prostecochloris aestuarii[(1983) Biochim. Biophys. Acta 725, 361–367]. It consists of 3 major polypeptides. The dominating one, of 65 kDa and carrying bacteriochlorophyll, is the P840 reaction center protein. It is probably very similar to the P700 reaction center protein of chloroplasts and cyanobacteria. A 24‐kDa protein could be identified as an ascorbate‐reducible cytochrome c‐550.5. The third polypeptide of 32 kDa might be a Rieske‐type FeS protein. The cytochrome bfraction consists of 2 polypeptides of 42 kDa, representing cytochrome b‐562, and 24 kDa, representing ascorbate‐reducible cytochrome c‐550.5 again. Conditions can be varied to obtain cytochrome b‐562 in pure form. A first characterization of the components is presented.

When the cytochrome b6/fcomplex from chloroplasts is incorporated into liposomes, reduction of external plastocyanin by plastoquinol is stimulated under uncoupling conditions. An extra H+/e−is ejected from the vesicles during the reaction, in addition to the scalar proton liberated from plastoquinol. This is stimulated by valinomycin/K+and abolished under uncoupling conditions. Furthermore, the formation of a membrane potential during the reaction, negative inside the vesicles, is observed with the help of carbocyanine dyes. We conclude that the cytochrome b6/fcomplex, like the cytochrome bc1from mitochondria, functions as an electrogenic proton translocator.

ABSTRACTPseudouridine (?) residues were localized in theSaccharomyces cerevisiaespliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiaeUsnRNAs contain only a few ? residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1,PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA ? content, only the loss of the Pus1p activity was found to affect ? formation in spliceosomal UsnRNAs. Indeed, ?44formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze ?44formation in the S. cerevisiaeU2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA ? content, formation of ? residues in S. cerevisiaeUsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.