Molecular cloning and analysis of the nuclear gene MRP-L6 coding for a putative mitochondrial ribosomal protein from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae nuclear gene MRP-L6 was cloned by complementation of the respiratory-deficient mutant pet-ts 2523 with a library of wild-type yeast genomic DNA. The isolated gene was part of a 3.8-kb sequenced DNA fragment containing, in addition to MRP-L6, two unassigned reading frames, ORF1 and ORF2. MRP-L6 codes for a basic protein of 205 amino acids and a molecular mass of 22.8 kDa. The protein exhibits significant sequence similarity to the ribosomal protein L6 of bacteria and chloroplasts. Unlike the corresponding bacterial proteins, however, the MRP-L6 protein (MRP-L6p) contains at its N-terminus a 16 amino-acid leader sequence exhibiting the known characteristics of mitochondrial import signals. Disruption of MRP-L6 leads to the phenotype of a mitochondrial translation-defective, rho-negative yeast mutant. The results are consistent with MRP-L6p representing an essential component of yeast mitochondrial ribosomes. Expression of MRP-L6 was examined, under conditions of glucose repression and derepression, in wild-type cells and in a series of catabolite repression-defective yeast mutants. In most cases, a distinct though small influence of the carbon source on the expression of an MRP-L6/lacZ reported construct was observed.

Nearest-neighbor analysis of a photosystem II complex from Marchantia polymorpha L. (liverwort), which contains reaction center and antenna proteins.

A new photosystem II preparation was isolated from Marchantia polymorpha thylakoids upon solubilization with dodecyl beta-D-maltoside and glycerol gradient ultracentrifugation. Its protein composition was analyzed, and all tested polypeptides from the core complex, the oxygen-evolving enhancer and the light-harvesting complex (LHC) could be detected. The only component severely depleted compared with the grana membrane preparation was the psbS gene product. This complex was subjected to chemical cross-linking using the cleavable homobifunctional cross-linker dithiobis(sulfosuccinimidylpropionate). The overall pattern of cross-linking-products was analyzed by diagonal electrophoresis, where the cross-linking agent was cleaved by reduction of the disulfide bond between the first and second dimensions of the gel, followed by immunoblotting. Many cross-linking products were characterized and these data used in order to identify protein masses revealed by electron microscopy [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179]. It is concluded that the core proteins CP43 and CP47 are located at opposite sides of the D1-D2-cytochrome b559 complex. Minor CAB proteins were found to interact with core complex subunits (CP29, CP26) and LHCII (CP26), supporting the view that these proteins could interface the major LHCII with the reaction center.