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.

Osh-dependent and -independent Regulation of PI4P Levels During Polarized Growth ofSaccharomyces cerevisiae

“…Yet, we also observed that Ypt32p localized to both vesicles and sites of polarized growth (i.e., bud tip and bud neck), suggesting Ypt32p is not lost from vesicles during trafficking, consistent with other studies ( Ling et al. , 2014 ; Gingras et al. , 2022 ).…”

“…This might occur when the vesicle is in close proximity to the PM rather than during trafficking in the mother cell. Our study and that of Gingras et al. (2022) , in which single vesicles are tracked, support such models.…”

“…Therefore, Ypt32p may only dissociate from vesicles just before vesicle docking at the PM. This idea however is inconsistent with the idea of a GTPase exchange during trafficking, especially in light of evidence for the presence of Sec4p at the Golgi ( Gingras et al. , 2022 ).…”

Use of Protein interactions from Imaging Complexes after Translocation (PICT) to characterisein situthe spatial configuration of proteins interacting with the exocyst

“…To be functional, the exocyst complex requires the binding of the Sec15 subunit to Sec2 ( 12, 26, 27 ). The spatial distribution of the 23 ± 9 copies of Sec2 that, on average, populate each secretory vesicle remains unknown ( 42 ).…”

Small GTPase Rab7 is involved in stress adaptation to carbon starvation to ensure the induced cellulase biosynthesis in Trichoderma reesei

“…Thereafter, transport vesicles originating from the Golgi deliver various cargo proteins to different destinations, including the organelles of secretory and endocytic pathways, the plasma membrane, and the extracellular space [ 9 , 10 ]. Therefore, the entire and highly ordered process of post-Golgi vesicle trafficking is finely regulated and coordinated by the Rab GTPases [ 11 ], which is the largest subfamily of small G proteins and regulates many trafficking events, including vesicle biogenesis, transport, tethering, docking, and fusion with target membranes [ 12 ]. Specifically, fusion of late endosomes or vesicles from TGN with vacuole is mediated by the small GTPase Rab7 and its effectors [ 13 , 14 ].…”