Figure legends

III. 5. Figure legends

Figure 17: Chimeric csA fusion proteins. A) Chimeric csA-SibA TM is composed of the csA extracellular domain fused to the TMD of SibA and to a short cytoplasmic tail. In csA-TM the extracellular domain of csA is fused to a 21-residue long hydrophobic TMD and to the same short cytoplasmic tail. B) Amino acids sequence of the transmembrane and cytoplasmic portions of the two constructs. The yellow and green boxes indicate the positions of the KpnI and XbaI restriction sites used to clone DNA fragments in the expression vector.

Figure 18: Immunofluorescence labeling of csA. The surface of live cells is labeled with an antibody against csA. Cells are then fixed and labeled with a secondary antibody coupled to an Alexa 647 fluorophore. Cells are then permeabilized and the same antibody against csA is applied followed by a secondary antibody coupled to an alexa 488 fluorophore. Cells are then analyzed by flow cytometry to measure simultaneously both fluorescence channels for each individual cell.

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Figure 19: Detection of csA surface expression by flow cytometry. Left panel: non-transfected WT cells were analyzed as a negative control. Right panel: a typical plot obtained with WT cells expressing csA-SibATM. Each dot corresponds to a single cell. The X axis (alexa 488) corresponds to intracellular fluorescence intensity and the Y axis to the fluorescence intensity detected at the cell surface.

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Figure 20: Analysis of cell surface localization of csA chimeras in WT and phg1A KO cells by flow cytometry. These graphics reveal the surface localization of two csA chimeras in two different cell types: WT and phg1A KO. The two upper panels show the distribution of csA-SibA TM in WT cells (left graphic) and in phg1A KO cells (right graphic). The two lower panels show the distribution of csA-TM in both cell types. Results indicate that a similar proportion of csA-TM construct is localized at the cell surface in WT and in phg1A KO cells, while the csA-SibA TM construct is more present at the cell surface in WT cells than in phg1A KO cells.

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Figure 21: Analysis of flow cytometry data. This scheme shows how the distribution of fluorescent intensities was analyzed. In order to extrapolate a numeric value from this plot, the graphic was divided in equivalent slices (grey). The X and Y mean values of each slice were calculated (squares on the right panel). A linear regression was used (red curve) and its equation is indicated. The general formula for each curve is y=Ax+B where A is the slope of the curve.

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Figure 22: Surface localization of csA-SibA TM and csA-TM in WT and phg1A KO cells. For each csA chimera, the slope obtained with the phg1A KO cells as described in Figure 17 was divided by the slope obtained with the WT cells. This graph shows average values of 14 experiments. The SEM is shown. Ratios obtained are significantly different (Student t test;

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Figure 23: Cell surface localization of csA-SibA TM visualized by immunofluorescence. WT and phg1A KO cells expressing a csA-SibA TM chimeric protein were immunolabelled with a monoclonal antibody against csA (41-17-21). The cell surface was labeled first, then after fixation and permeabilization, the intracellular csA was labeled. The cell surface staining was revealed with Alexa647-labeled secondary antibodies, the intracellular staining with Alexa 488-labeled antibodies.

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Figure 24: Cell surface localization of csA analyzed by cell surface biotinylation. This scheme summarizes how cell surface biotinylation was used to quantify the proportion of csA present at the cell surface. First, all proteins present at the cell surface were labeled with NHS-SS biotin. Cells were then lysed and an aliquot collected to evaluate the total amount of cellular csA. Biotinylated proteins were isolated on neutravidin beads, then detached from beads separated on SDS-PAGE gels. Chimeric csA proteins were detected using a specific monoclonal antibody (33-294-17).

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Analysis of csA total (anti-csA)

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Figure 25: Surface and total csA analysis following cell surface biotinylation. A) Gels obtained at the end of the biotinylation assay as depicted on figure 24. Bands correspond to the indicated csA chimeras. B) Band intensities were measured, allowing quantifying the surface and the total csA expression. The percentage of the total csA present at the cell surface of each cell type was calculated. SEM are shown. (Student t-test csA-SibA TM p<0.001, Student t-test csA-TM p=0.48)

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Figure 26: Mature csA exhibits two carbohydrates. A) Schematic view of csA maturation after synthesis in the ER. A first carbohydrate (CH I) is cotranslationaly added to the protein in the ER.

A second carbohydrate, CH II, will be added in the Golgi. Mature csA has an apparent molecular weigh of 80 KDa, while the partially glycosylated form has an apparent molecular weight of 68 KDa. B) Mature csA can be detected with the antibody 12-120-94 directed against CH II, while the antibody 33-294-17, which binds to the protein moiety will recognize both csA glycosylated forms.

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Figure 27: csA-SibATM and csA-TM exhibit similar stability in WT and phg1A KO cells.

Stability of csA-SibA TM and csA-TM was assessed in WT and phg1A KO. Cycloheximide, an inhibitor of protein synthesis, was added to the culture medium. Aliquots of the cell suspension were taken after 0, 2 and 4 hours. After SDS-PAGE electrophoresis, csA proteins were detected with the antibody 33-294-17. The graphics indicate the intensities of the high- and low-molecular weight csA forms. Full bars correspond to the upper csA band and empty bars are the lower band.

A) Stability of csA-SibA TM (n=5). B) Stability of the csA-TM constructs (n=3). The variation with time of the total amount of csA (lower + upper bands) for both constructs indicates that proteins are stable in WT and phg1A KO cells.

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Fig. 28: The SibA transmembrane domain exhibits a glycine motif. The amino acid sequence of the putative transmembrane domain of SibA reveals the presence of four glycine residues (in red) arranged on the same face of the TM helix as represented schematically.

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Figure 29: The cell surface localization of glycine-rich TM domains depends on Phg1A.

A) Representation of csA chimeric constructs. The csA-G3 chimera is a synthetic construct made of the csA extracellular domain fused to a transmembrane domain containing four glycine residues and a short cytoplasmic tail. In the csA-H0 chimera, the glycine residues are replaced with hydrophobic residues. B) Amino acid sequence of the transmembrane and cytoplasmic domains of both constructs. C) Flow cytometry analysis of WT and phg1A KO cells transfected with these two constructs, as detailed in the legend to figure 18 reveals that cell surface expression of csA-G3, but not csA-H0, is dependent.on Phg1A (n=10) (t-test, p<0.01).

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Figure 31: Assessing interaction between Phg1A and Tac chimeric proteins. This scheme summarizes the assay performed to reveal a putative association of Phg1A with glycine-rich transmembrane domains. After cotransfection of Hela cells with plasmids encoding Phg1A protein fused to $-Galactosidase and a single pass transmembrane protein with the Tac extracellular domain.

Cell lysates were incubated with beads coated with an anti-Tac antibody. Interaction of Tac TMD with Phg1A is revealed by measuring $-Galactosidase activity with CPRG.

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