Large-Scale Analysis of Membrane Transport in Yeast Using Invertase Reporters

Abstract

Transport of membrane proteins between cellular organelles requires the concerted action of many regulatory factors, which aid in cargo recognition and vesicle formation, targeting, and fusion. The yeast Saccharomyces cerevisiae is a useful model system for studying such regulators, due to the availability of genome-wide mutant collections and reporter proteins that provide sensitive biochemical readouts of individual transport pathways. Here, we describe an enzymatic invertase assay for evaluating endocytic recycling using a chimeric GFP-Snc1-Suc2 reporter. Cell surface levels of this reporter can be measured by a colorimetric assay that monitors sucrose hydrolysis at the plasma membrane, using two different methods. The first is a semiquantitative agar overlay assay followed by image densitometry that is suitable for high-throughput screening of arrayed yeast colonies. In the second, more quantitative assay, an enzymatic solution is added to yeast cultures in a multi-well plate and the absorbance is assessed by a plate reader. Furthermore, the modular nature of the chimeric reporter allows alternate transport signals to be introduced, thereby expanding the range of transport pathways that can be evaluated by this method. Together these techniques can be used to explore the function of genes involved in a variety of cellular trafficking pathways.

Cargo selectivity of yeast sorting nexins

“…The colorimetric liquid invertase assay used here is based on the assays described by Darsow et al and Dalton et al First, overnight cultures grown in synthetic selective media with 2% fructose were diluted to 1 OD 600 or 10 OD 600 /mL depending on the expected level of secretion. A total of 20 μL of the stocks were added to 30 μL of 0.1 M sodium acetate.…”

The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

“…The identification of Apm2, but not the more abundant μ-subunit isoform Apm1, was unexpected and suggested that Apm2-containing AP-1R complex has a specific role in Snc1 sorting. To confirm these observations, we used a quantitative assay that measures the invertase (Suc2) activity of GSS present at the cell surface ( Figure 1A ; Dalton et al. , 2015 ).…”

“…Strains expressing the GSS construct were grown for 20 h in 2 ml of YP-fructose (final OD 600 of ∼10). Cultures were diluted to 3 OD 600 /ml, and the liquid invertase assay was performed as described ( Dalton et al. , 2015 ).…”

Genetically encoded multimode reporter of adaptor protein 3 (AP-3) traffic in budding yeast

“…resulting reporter, GSS (here called GSI; Figure 1A) was used to assay the integrated levels of endocytosis and exocytosis in high-throughput screens of yeast gene deletion mutants. [16][17][18] The v/R-SNARE protein Nyv1 is normally targeted to the limiting membrane of the yeast vacuole through the AP-3 pathway. 19,20 Nyv1 sorting requires interaction between a YXXΦ-like motif (YGTI) in Nyv1's N-terminal longin domain and Apm3, the AP-3 μ-chain.…”

“…Overall, these results indicate that GNSI is suitable for large-scale screening using automated mating and sporulation procedures. 17,26 Finally, we asked if GNSI could be used in growth selections. Mutants lacking extracellular invertase exhibit growth defects when sucrose rather than dextrose is the primary carbon source.…”