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Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation
X Foissac1, C Saillard, J Gandar
1Laboratoire de Biologie Cellulaire et Moléculaire, Institut National de la Recherche Agronomique and Université de Bordeaux II, Villenave d'Ornon, France.
Abstract:
Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.
Insights
Polymorphism in Spiroplasma citri spiralin, a major membrane protein, is not caused by palmitic acid modification. Differences in spiralin structure, not post-translational modifications, likely explain mobility variations in this mollicute.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Chemistry
Background:
- Spiralin is the primary membrane protein in Spiroplasma citri, a helical mollicute.
- Observed variations in spiralin's electrophoretic mobility depend on the S. citri strain.
- Understanding spiralin's structural basis for mobility differences is crucial for mollicute research.
Purpose of the Study:
- To investigate the molecular basis of spiralin polymorphism in Spiroplasma citri.
- To determine if post-translational modifications, specifically palmitoylation, contribute to spiralin's electrophoretic mobility variations.
- To elucidate the structural properties underlying spiralin's strain-specific characteristics.
Main Methods:
- Sequencing of PCR-amplified spiralin genes from eight S. citri strains.
- Analysis of protein molecular masses derived from gene sequences.
- Labeling of S. citri proteins with [3H]palmitic acid and treatment with globomycin.
- Formic acid hydrolysis of labeled spiralin followed by electrophoretic analysis of peptide fragments.
Main Results:
- Spiralin gene sequences revealed consistent lengths (241 or 242 amino acids), with conserved N-terminal signal peptides.
- Calculated molecular masses did not correlate with observed electrophoretic mobility differences.
- Palmitoylation was confirmed for spiralin and other proteins, but hydrolysis experiments indicated palmitic acid modification occurred on a conserved fragment.
- Differences in electrophoretic mobility of whole spiralin proteins were significantly larger than those of their peptide fragments after hydrolysis.
Conclusions:
- Spiralin polymorphism in S. citri is primarily a structural property of the protein itself, not due to variations in palmitoylation.
- The observed mobility differences likely stem from inherent structural variations within the spiralin protein.
- An unidentified post-translational modification could potentially contribute to spiralin polymorphism, though structural differences are the most probable cause.