RESUMO
The alkaline phosphatase signal peptide participates in transport of the enzyme to the periplasmic space of Escherichia coli. The signal sequence, like that of other signal peptides, is composed of a polar amino-terminal segment, a central region rich in hydrophobic residues and a carboxy-terminal region recognized by signal peptidase. We have previously shown that an alkaline phosphatase signal peptide mutant containing a polyleucine core region functions efficiently in transport of the enzyme [D. A. Kendall, S. C. Bock, and E. T. Kaiser (1986) Nature 321, 706-708]. In this study, some of the amino acid changes involved in the polyleucine sequence are examined individually. A Phe to Leu substitution as the sole change results in impaired transport properties in contrast to when it is combined with three other amino acid changes in the polyleucine-containing sequence. A mutant with a Pro to Leu substitution in the hydrophobic core region is comparable to wild type while the same type of substitution (Pro to Leu) in the carboxy-terminal segment results in substantial accumulation of the mutant precursor. Finally, introduction of a basic residue into the hydrophobic segment (Leu to Arg substitution) results in a complete export block. These results exemplify the spectrum of properties produced by individual residue changes and suggest there is some interplay between hydrophobicity and conformation for signal peptide function.
Assuntos
Fosfatase Alcalina/metabolismo , Aminoácidos/metabolismo , Escherichia coli/enzimologia , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutação , Sinais Direcionadores de Proteínas/genéticaRESUMO
A systematic series of mutants has been generated which provides a means for titrating the dependence of protein transport activity on signal peptide hydrophobicity. These mutants involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide while maintaining the natural amino- and carboxyl-terminal segments and the overall length. The new core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine. Using precursor processing to quantify transport activity, we observe a clear, nonlinear dependence on hydrophobicity. At ratios of alanine to leucine of less than or equal to 8:2, the signal peptide is essentially nonfunctional; at ratios greater than or equal to 3:7, the signal peptide functions efficiently. The midpoint is between alanine to leucine ratios of 6:4 and 5:5. Signal peptides with hydrophobicity just below the midpoint show substantial, additional precursor processing over time while the others do not. The data are consistent with a simple model involving a two-state equilibrium between the untransported and transported species and a change in the delta G of -0.85 kcal/mol for every alanine to leucine conversion.
Assuntos
Fosfatase Alcalina/genética , Escherichia coli/genética , Metionina/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sequência de Bases , Transporte Biológico , Calorimetria , Escherichia coli/enzimologia , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Conformação Proteica , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Mapeamento por RestriçãoRESUMO
Cardiac hypertrophy is characterized, among others, by the molecular events which selectively activate the expression of genes for contractile proteins within individual myocardial cells. As such, myosin light chain 2 (MLC-2), which is upregulated in the hypertrophic state in both rat and human, serves as a marker for hypertrophy. In an attempt to investigate the gene regulatory mechanisms of this phenomenon, we tested the hypothesis that certain transcription factors are directly involved in the development of cardiac hypertrophy by demonstrating the presence of cardiac tissue-specific regulatory elements in the 5'-flanking region of the MLC-2 promoter and testing them in the gel mobility shift assay for their binding activity to nuclear proteins from hypertrophied and normal cardiac tissue. In nuclear extracts from the ventricular tissues of the spontaneously hypertensive rat (SHR), distinctive changes in two families of activator proteins, the A/T-rich DNA-binding transcription factors, myocyte enhancer factor (MEF-2) and CArG-binding factor, manifested in a developmentally dictated manner paralleling the evolution of cardiac hypertrophy in these animals. Extracts isolated from brains and skeletal muscle tissues from the same animals did not exhibit the changes in binding activity. Also, the changes were not apparent when a distal negative regulatory element (CSS), which confers cardiac-specific expression, was tested in gel mobility shift assays. The ubiquitous TATA-binding proteins remained unchanged in comparing SHR with the control strain WKY in the same assay. These data support the notion that the expression of specific transcription factors is modulated in response to hypertrophy related signals which execute changes at the gene level effecting the enrichment of certain contractile proteins in an effort discrete and estranged from the basal transcription machinery.