High-resolution secretory timeline from vesicle formation at the Golgi to fusion at the plasma membrane in S. cerevisiae

Abstract

Most of the components in the yeast secretory pathway have been studied, yet a high resolution temporal timeline of their participation is lacking. Here we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4's GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 seconds prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange.

Generation and characterization of conditional yeast mutants affecting each of the two essential functions of the scaffolding proteins Boi1/2 and Bem1

“…To assess relative dynamics of Boi1/2 patches on the membrane, the lifetime of single patches was measured (Figure 1F and G, Video 1 and 2). Boi1 patches were found to be shorter-lived with a mean lifetime of about 9s, whereas Boi2 patches persisted longer, with a lifetime of about 14s, a timing consistent with earlier measurements (Figure 1H) (Gingras et al, 2022). To explore whether Boi1 and Boi2 patches colocalize, cells were imaged expressing Boi1-mNG together with Boi2-mScarlet.…”

“…Strikingly, the majority of Boi2-mNG patches do not colocalize with secretory vesicles, or vice versa, although some colocalization of tethered vesicles and Boi2 patches does occur (Figure 3F, Video 4). The majority of non-colocalization is at least partially due to the relatively short tether-to-fusion time of secretory vesicles and the number of untethered vesicles in the bud (Gingras et al, 2022). Furthermore, examination of videos shows that secretory vesicles tether in places apparently devoid of Boi2-mNG patches.…”

“…Our analysis found that Boi1/2 remains at the cell cortex upon shifting sec6-4 to the restrictive temperature, again suggesting that they may function upstream of exocytosis. We also asked if Boi1/2 patches correlate with sites of secretory vesicle exocytosis especially given that Boi1/2 patches have a lifetime about double that of secretory vesicle tethering (Gingras et al, 2022). Interestingly, there was very little colocalization between Boi1/2 and Sec4, and since Boi2 patches do not move extensively on the membrane, it is difficult to reconcile with a model that invokes a direct and essential role for Boi1/2 in the tethering/exocytosis of secretory vesicles.…”

“…The actin cables are then used as the tracks for the myosin-V Myo2 to deliver post-Golgi secretory vesicles, marked by the Rab Sec4, to sites of exocytosis (Kabcenell et al, 1990; Pruyne et al, 1998; Salminen & Novick, 1987). During transport, the secretory vesicles acquire the exocyst, a multi-protein complex that facilitates the tethering of secretory vesicles to the plasma membrane and is essential for fusion with the plasma membrane in a process also involving Cdc42 (Gingras et al, 2022; TerBush et al, 1996; Wu et al, 2010).…”

Exocyst functions in plants: secretion and autophagy

“…This also means that, in plants, membrane bound exocyst holocomplex is possibly strongly stabilized compared to the free cytoplasmic form [5,12,26]. Yeast holocomplex assembles on the TGN‐derived exocytotic vesicles [35,36] and published observations and known interactions within plant complex subunits imply a similar possibility also for plants, at least in some cell types [22,23,26]. Interestingly, there are some subunit interactions that are common to animals and plants, and absent in yeast: SEC6, SEC8 and SEC10 interactions with SEC3; SEC15 interactions with the EXO70 and EXO84 (Fig.…”