Figure 1. Lipoprotein AmyE is triacylated.

A. AmyE localization in C. glutamicum wild-type cells expressing AmyE (pAmyE) or a variant of AmyE with a point mutation substituting the cysteine +1 by a leucine (pAmyE^C1L). An empty vector (pCGL482) was used as a control. Membrane and secreted proteins were analyzed by SDS-PAGE followed by immunoblotting using monoclonal anti-his antibodies. The band labeled with an asterisk corresponds to a shorter form of AmyE. B. AmyE (left panel) and AmyE^C1L (right panel) were purified and analyzed by SDS PAGE before (lane 1) or after Triton X114 extraction. Proteins from both aqueous (lane 2) and detergent (lane 3) phases were precipitated and loaded on the gel. C. MALDI mass measurements of intact purified AmyE and AmyE^C1L proteins. Estimated mass accuracy is 150 Da. D. MALDI PMFs of AmyE and AmyE^C1L proteins purified from C. glutamicum wild-type strain. The m/z 3200–4800 region of the mass spectra of AmyE and AmyE^C1L tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are indicated. Upper panel: m/z 4097.10 (bold) corresponds to the triacylated AmyE_1–29 peptide while m/z 3451.65, 3821.53 and 4045.88 match to internal tryptic peptides, AmyE_334–365, AmyE_366–397 and AmyE_55–91 respectively. m/z 4061.74 and 4077.54 peaks could correspond to mono- and di- oxidized AmyE_55–91 peptides. Bottom panel: m/z 3292.60 (bold) corresponds to the C1L-mutated AmyE_1–29 peptide while m/z 3451.65 and 3821.72 match to AmyE_334–365 and AmyE_366–397 peptides. The m/z 4061.79, 4077.80, 4093.86 and 4109.83 peaks match to mono-, di-, tri- and tetra-oxidized AmyE_55–91 peptides. The m/z 4342.89 peak corresponds to the di-oxidized AmyE_277–315 peptide. It’s worth noting that AmyE^C1L peptides were more often detected in the oxidized state than AmyE peptides. Insets aim at emphasizing the specificity of the m/z 4097.10 signal detected only in the AmyE spectrum. Asterisks indicate that m/z assignments are not accurate because of low mass resolution and weak signal/noise ratio. E. Sequence of the recombinant purified wild-type AmyE protein. Identified unmodified peptides after trypsin digestion and DDM/CHCl₃-CH₃OH treatment are shown in boldface type on the amino acid sequence of recombinant AmyE.