Functional distribution of the candidate palmitoylated proteins.

<p>The 93 proteins meeting the determined criteria for being candidates (including known palmitoylated proteins, but depleted from MHC molecules) were classified according to the biological processes they are associated with, as described in the Human Protein Reference Database. The “transport” category has been further manually subdivided in “molecular transport” and “vesicular transport” categories.</p

Graphical analysis of the B cell palmitoyl-proteome.

<p>(A) Overlap between proteins identified in the HA− or HA+ samples. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037187#s2" target="_blank">Results</a> from the triplicate analyses were merged and led to a list of 493 proteins that distribute as depicted in the Venn diagram. The numbers of proteins identified exclusively in the HA− samples, common to HA− and HA+ samples or exclusively identified in the HA+ samples are indicated. (B) Proteins identified in the HA+ samples (452 proteins) were plotted by their averaged normalized spectral counts in the HA+ (x axis) and the HA− (y axis) samples. The inset shows an expanded view of the lower values zone. Novel candidate palmitoylated proteins (•) cluster along the x-axis, as do established palmitoylated proteins (▴) and many candidate palmitoylated proteins identified in previous proteomic studies (▪). ○, proteins that were not significantly enriched, according to our statistical analysis; •, CD20 and CD23.</p

The immune regulators CD20 and CD23 are novel palmitoylated proteins.

<p>HEK293T cells were transfected with plasmids encoding His-tagged versions of flotillin-1, CD20, CD23 and Cys-to-Ala mutants of CD20 and CD23, and metabolically labeled with 17-ODYA. Cell membranes were then subjected to click chemistry prior to separation of proteins by SDS-PAGE on a 10% acrylamide gel. Proteins were treated (lower panels) or not (upper panels) with hydroxylamine before gel loading. Left panels: Scans of the fluorescent proteins. Right panels: Immunodetection of the exogenous proteins with an anti-His antibody. Arrowheads indicate the position of overexpressed proteins. The figure is a composite of two gels, as shown by a thick line. Lanes separated by a thin line were not originally contiguous in the gel, but were placed next to each other in the figure for the sake of clarity. Gels and blots were cropped around the region of interest.</p

List of the peptides allowing the identification of CD20 and CD23.

<p>List of the peptides allowing the identification of CD20 and CD23.</p

Palmitoylated proteins from human B lymphoid cells are enriched following ABE purification.

<p>Proteins from B lymphoid cells membranes were subjected to ABE purification with (HA+) or without (HA−) hydroxylamine treatment, resolved by SDS-PAGE on a 4–12% gradient gel, and detected by silver nitrate staining.</p

Schematic representation of CD20 and CD23.

<p>Cysteines that are potential sites of palmitoylation are indicated, as well as N and C termini. For the sake of simplicity, CD23 is depicted as a monomer, although CD23 molecules are assembled in trimers in the membrane <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037187#pone.0037187-Kilmon1" target="_blank">[36]</a>. The CD20 scheme is adapted with permission from Ernst <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037187#pone.0037187-Ernst1" target="_blank">[35]</a>. Copyright (2005) American Chemical Society.</p

Spectral index distribution of the identified proteins.

<p>The distribution of the SpI of the 353 proteins identified in both HA− and HA+ samples is represented as an histogram (medium grey). The random distribution of the SpIs was calculated according to Fu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037187#pone.0037187-Fu1" target="_blank">[32]</a>, in order to determine the SpI thresholds corresponding to the 5% and 1% confidence intervals (respectively 0.54 and 0.74), and is shown in the background (light grey). The distribution of the known palmitoylated proteins identified in this study is superimposed in black.</p

Pycnosomes present in <i>D</i>. <i>discoideum</i> endocytic compartments are secreted in the extracellular medium.

<p><i>D</i>. <i>discoideum</i> cells grown in axenic medium were fixed and processed for electron microscopy. (A) Most endocytic compartments appeared empty or contained occasionally a few vesicles (arrowhead). (B) Dense bodies (pycnosomes) appeared as amorphous structures in the endosomal lumen (star). (C) Secreted pycnosomes were recovered from the extracellular medium by differential centrifugation. Bars: 500 nm.</p

Electron microscopy reveals SctA-enriched endosomal pycnosomes.

<p><i>D</i>. <i>discoideum</i> cells grown in axenic medium were processed for immuno-electron microscopy. (A-B) Sections were labeled with the H161 anti-p80 antibody. The p80 protein was abundantly present in endosomal membranes, and only small amounts of p80 were found associated with pycnosomes (stars) in the lumen. (C-D) The B4.2 antibody revealed a high concentration of SctA in endosomal pycnosomes. In some pictures, pycnosomes appeared associated with some membranous elements (arrowheads). Bar: 500 nm.</p

SctA is a major constituent of secreted dense bodies.

<p>(A) <i>D</i>. <i>discoideum</i> cells were pelleted by centrifugation at 600 x g (pellet P6) and the supernatant (S6) was sequentially subjected to centrifugations at 15’000 and 100’000 x g. The corresponding supernatants (S15, S100) and pellets (P15, P100) resuspended in an equivalent volume were diluted in reducing sample buffer and separated on a 15% acrylamide gel. Equal volume of samples were loaded except for P6 that was diluted 1/10. The main proteins were revealed by silver staining. (B) The same amount of the 15 000 x g pellet was resuspended in reducing (+DTT) or non reducing (-DTT) sample buffer and analyzed by SDS-PAGE and Coomassie staining. Molecular weights (in kDa) are indicated, as well as the SctA protein (arrowheads).</p