Figure 4. Sec7-dependent cargo sorting events are sequential and coincide with the peak of Ypt31 levels.
(A) Live-cell imaging of GFP-Sec7-labeled TGN compartments disintegrating into smaller, fast-moving structures.
(B) Timelapse subseries (1 sec intervals) of the box from (A), a single Golgi compartment.
(C) Normalized quantification of the GFP-Sec7 signal in the box from (A).
(D) Timelapses (5 sec intervals) from strains expressing Sec7-6xDsRed and different Sec7-dependent cargos, aligned by measured Sec7 disappearance time. A strain expressing both GFP-Sec7 and Sec7-6xDsRed serves as a control.
(E) Timelapses (1 sec intervals) from strains expressing Sec7-6xDsRed and GFP-Ypt1 or GFP-Ypt31.
(F) Quantification of the peak-to-peak times. Error bars represent s.e.m. for n=5.
(G) Quantification of the relative disappearance time for cargos. Error bars represent s.e.m. for n=4 to 6.
(H) Model for the dynamics of Sec7-dependent events at the late Golgi. Sec7 disappearance is set to t=0. Peak times and sorting times are set relative to t=0. Sorting times denote when >80% of the cargo has exited the TGN. For simplicity, a single time (−11 s) is used to denote sorting of Kex2 (−13.5 s) and Tlg1 (−9 s). Times are denoted as approximate (~) when derived from two sequential relative comparisons. For example, the timing of Sec7 peak levels was measured relative to Sec7 disappearance (Figure S3E), while the peak levels of the Rab proteins were measured relative to the peak of Sec7 (Panel F). Dotted lines and arrows represent events not measured, but inferred from this study and others, for example, the timing of PI(4)P appearance relative to Sec7 (Ortiz et al., 2002; Daboussi et al., 2012). We envision that Sec7 must leave the membrane surface of a secretory vesicle (perhaps triggered by inactivation of Arf1 and Arl1 by the Gcs1 ArfGAP) before Ypt31/32 can recruit Sec2, leading to Sec4 activation and recruitment of the Myo2 (Myosin V) motor.