RESUMO
The integral membrane protein cytochrome f contains an amino-terminal signal sequence that is required for translocation into the thylakoid membrane. The signal sequence contains a hydrophobic core neighbored by an amino-terminal charged residue. Mutations that introduce charged amino acids into the hydrophobic core are inhibitory to cytochrome f translocation, and thus render cells non-photosynthetic. We have isolated both nuclear and chloroplast suppressors of these mutations by selecting for restoration of photosynthetic growth of Chlamydomonas. Here we describe the characterization of two chloroplast, second site suppressor mutations. Both suppressors remove the positively charged amino acid that borders the amino terminus of the hydrophobic core, and replace this arginine with either a cysteine or a leucine. The existence of these suppressors suggests that the hydrophobic core can be shifted in position within the signal sequence, and analysis of triple mutants in the signal confirms this hypothesis. Thus this signal that mediates translocation into the thylakoid membrane is characterized by a hydrophobic region whose exact amino acid content is not critical, and that need not be flanked on its amino terminus by a charged residue.
Assuntos
Chlamydomonas/metabolismo , Cloroplastos/metabolismo , Citocromos/química , Citocromos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Chlamydomonas/química , Chlamydomonas/genética , Citocromos/genética , Citocromos f , Eletroquímica , Proteínas de Membrana/genética , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/metabolismo , Supressão GenéticaRESUMO
We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.