Visualizing Secretory Cargo Transport in Budding Yeast

Abstract

Budding yeast is an excellent model organism for studying the dynamics of the Golgi apparatus. To characterize Golgi function, it is important to visualize secretory cargo as it traverses the secretory pathway. We describe a recently developed approach that generates fluorescent protein aggregates in the lumen of the yeast endoplasmic reticulum and allows the fluorescent cargo to be solubilized for transport through the Golgi by addition of a small-molecule ligand. We further describe how to generate a yeast strain expressing the regulatable secretory cargo, and we provide protocols for visualizing the cargo by 4D confocal microscopy and immunoblotting. C 2018 by John Wiley & Sons, Inc.Keywords: cargo r cisternal maturation r fluorescence microscopy r Golgi r yeast

A microscopy-based kinetic analysis of yeast vacuolar protein sorting

“…To create a wave of fluorescent vacuolar cargo, we modified a recently developed regulatable fluorescent secretory cargo ( Casler and Glick, 2019 ; Casler et al, 2019 ). Our secretory cargo consists of the tetrameric red fluorescent protein DsRed-Express2 fused to a dimerizing variant of FKBP, with a cleavable N-terminal signal sequence to direct cotranslational translocation into the ER.…”

“…AP-1 was originally proposed to mediate transport of proteins from the TGN to endosomes (reviewed in Hinners and Tooze, 2003 ), but subsequent work implicated GGAs in TGN-to-endosome traffic in both yeast and mammalian cells ( Black and Pelham, 2000 ; Dell'Angelica et al, 2000 ; Hirst et al, 2000 ; Zhdankina et al, 2001 ). In S. cerevisiae , AP-1 localizes exclusively to the late Golgi, and it mediates intra-Golgi recycling of some resident late Golgi proteins and secretory cargoes ( Casler and Glick, 2019 ; Day et al, 2018 ; Liu et al, 2008 ; Papanikou et al, 2015 ; Spang, 2015 ; Valdivia et al, 2002 ). It was previously reported that yeast GGAs function upstream of AP-1 and that GGAs display similar kinetics of arrival and departure as the late Golgi reference marker Sec7 ( Daboussi et al, 2012 ).…”

“…Live-cell fluorescence microscopy complements other methods by providing unique insights into membrane traffic ( Lippincott-Schwartz et al, 2000 ). Recently, we engineered a regulatable fluorescent secretory cargo that can be visualized during yeast Golgi maturation ( Casler and Glick, 2019 ; Casler et al, 2019 ). This secretory cargo is present in Golgi cisternae throughout the maturation process, and a fraction of the cargo molecules recycle from older to younger cisternae by an AP-1-dependent pathway ( Casler et al, 2019 ).…”

“…Such experiments require a way to visualize the transport of a biosynthetic vacuolar cargo in live yeast cells. To this end, we built on our recent engineering of a regulatable fluorescent secretory cargo ( Casler and Glick, 2019 ; Casler et al, 2019 ). A tetrameric red fluorescent protein is fused to an improved dimerizing variant of the FK506-binding protein FKBP, and this construct is targeted to the ER lumen to generate aggregates, which are then solubilized with a ligand to create a fluorescent cargo wave that passes through the Golgi.…”

Maturation-driven transport and AP-1–dependent recycling of a secretory cargo in the Golgi

“…This approach required overcoming a series of technical hurdles to ensure that the fluorescent secretory cargo reaches the ER lumen, that the cargo aggregates dissolve efficiently, and that the cargo traverses the secretory pathway without being diverted to the vacuole (Casler and Glick, 2018). A key step was to add a tripeptide ER export signal to the cargo (Nam et al, 2014; Yin et al, 2018).…”