Figure 5. Sec5 phosphorylation contributes to a cyclic regulatory loop upon recognition by RalA.

(a) Sec5 Ser89 phosphorylation is concentrated at the abscission site during cytokinesis. Cos-1 cells were stained with antibodies against Sec8 (green) and p-Ser89 Sec5 (red), and subjected to confocal microscopy. When indicated, cells were stained with equal amount of antibodies plus p-Ser89 antigenic peptide. Images were at the same magnification; bar = 10μm. (b) Structural illustration of spatially positioning of Ser89 in Sec5 RBD. Figures were generated from the structure of Sec5 Ral binding domain (PDB ID: 1HK6) using Pymol software (DeLano Scientific LLC). (c) D⁷³S substitution alleviates the inhibitory effects on RalA interaction observed with S⁸⁹D RBD. Lysates of Cos-1 cells expressing indicated constructs were incubated with immobilized GST-RalA G²³V. RBD bound to RalA or present in total cell lysates was determined by WB. (d) Reconstitution of phosphorylation-dependent disengagement between RalA and Sec5. Upper: Schematic of the hypothetic sequential events during phosphorylation-induced disengagement. Lower: A complex between recombinant His-RalA loaded with GTPγS and GST-Sec5 RBD was incubated with PKC, and was immobilized by GSH column. RalA bound to Sec5, or released into supernatant, was determined by WB. (e) Loss of Sec5 phosphorylation leads to accumulation of binding with endogenous RalA. Cos-1 cells expressing the indicated constructs were stimulated as indicated before being subjected to anti-HA IP. Bound RalA were detected by WB using protein-A conjugated secondary antibody. (f) Total internal reflection fluorescent microscope (TIRFM) to evaluate the role of Sec5 phosphorylation. Representative images from Cos-1 cell over-expressing pHluorin-Transferrin receptor (TfR) and wild type Sec5 (upper left) or Sec5 S/A (lower left). Scale bar = 10μm. Representative fusion events marked by red boxes 1 & 2, or red boxes 3 & 4. Upper right, time series images of sample fusion events (1 & 2) for Sec5 WT and (3 & 4) for Sec5 S/A. Each panel = 5μm. Lower right, pHluorin-TfR fusion events per area (μm²) per time (min) observed in Sec5 WT and Sec5 S/A Cos cells. Over 1500 fusion events from three different experiments (n=3) were quantified. Errors represent SD. Asterisk: p<0.05.