Figure 4

Secretion of p17 does not occur if its W36 residue is mutated or when it is expressed alone.

(A) Quantification of extracellular p17 released by Jurkat cells expressing W16A or W36A Gag mutants. Cells were nucleofected with construct expressing wild type Gag or Gag mutants containing W16A and W36A amino acid changes. After 24 h later cells were cultured for 16 h onto ELISA plates and secreted p17 was quantified by cellular ELISA. Box plots represent statistical analyses of five independent experiments. Significance was assessed using one-way ANOVA and the Bonferroni’s post-test was used to compare data; ***P < 0.001. (B) Confocal analysis of Gag localization. HeLa nucleofected with the same constructs as in (A) were stained with biotinylated anti-p17 mAb MBS-3 followed by Alexa488-streptavidin (green) and 4′,6-diamidino-2-phenylindole (in blue). Representative median confocal sections of at least 20 cells per conditions are shown. Images highlight that W36A mutation leads to protein accumulation in proximity of the plasma membrane. z-Stack sections and orthogonal z reconstitution are also shown. Scale bar, 10 μM. (C) Quantification of extracellular p17 released by Jurkat cells expressing wild type p17 (myr⁺) or its G2A mutant (myr⁻). Cells were nucleofected with the construct that expresses wild type Gag, or with constructs that express p17 or a mutant containing the G2A amino acid change (p17 G2A). Cells were recovered 24 h later, then cultured for 16 h onto ELISA plates and secreted p17 was quantified by cellular ELISA as in Fig. 1. Box plots represent statistical analyses of five independent experiments. Significance was assessed using one-way ANOVA and the Bonferroni’s post-test was used to compare data; ***P < 0.001. (D) Confocal analysis of p17 localization. HeLa nucleofected with the same constructs as in (C) were stained with biotinylated anti-p17 mAb MBS-3 followed by Alexa488-streptavidin (green) and 4′,6-diamidino-2-phenylindole (in blue). Representative median confocal sections of at least 20 cells per conditions are shown. Images highlight that p17 alone is trapped in the cell cytosolic compartment regardless of whether or not it is myristoylated. z-Stack sections and orthogonal z reconstitution are also shown. Scale bar, 10 μM.