Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum.
The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [35S]cysteine, [35S]methionine, [14C]myristic acid (14C-14:0), [14C]palmitic acid (14C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.
Le Hénaff
M
M
Université de Rennes 1, Groupe Membranes et Osmorégulation, UPRES-A CNRS 6026, France. m-lehenaff@enitab.fr
Fontenelle
C
C
D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D003470:Culture Media; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D013149:Spiroplasma / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000378:metabolism
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D003470:Culture Media; D005227:Fatty Acids; D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; C028588:spiralin; C031164:glycerylcysteine; D003545:Cysteine