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. © 2018 by John Wiley & Sons, Inc.

Keywords: Golgi; cargo; cisternal maturation; fluorescence microscopy; yeast.

Figure 1.. A reversibly aggregating fluorescent secretory cargo.

(A) Strategy for generating and dissolving fluorescent aggregates. DsRed-Express2 tetramers (red) fused to a dimerizing variant of FKBP (gold) associate with one another to form aggregates. Addition of the FKBP ligand SLF (blue) blocks dimerization, thereby dissolving the aggregates into soluble tetramers. (B) Schematic of the functional segments of the regulatable secretory cargo polypeptide. (1) pOst1 (green): ER signal sequence that directs cotranslational translocation. (2) APV (pink): ER export signal tripeptide. (3) NTT (blue): N-linked glycosylation signal tripeptide. (4) DsRed-Express2 (red): tetrameric fluorescent protein. (5) FKBP^RD(C22V) (gold): reversibly dimerizing variant of FKBP. The lengths of the different segments are not to scale.

Figure 2.. Visualizing the secretory cargo together with Golgi markers.

A strain expressing pOst1-APVNTT-DsRed-Express2-FKBP^RD(C22V), GFP-Vrg4 (early Golgi marker), and Sec7-HaloTag (late Golgi marker) was grown to mid-log phase in NSD, labeled with the HaloTag-JF₆₄₆ dye (Grimm et al., 2015), washed, mounted in a flow chamber, and imaged by 4D confocal microscopy while SLF was flowed over the cells. Images, taken from Movie 1, are average projected Z-stacks at the indicated time points. Scale bar, 2 μm.