A high-content quantitative imaging screen identifies regulators of the post-Golgi transport of the phospholipid flippase Neo1. Neo1 is a new Snx3 cargo protein that is required for sorting of other Snx3 cargo. Thus flippase activity may generate curvature to promote the formation of Snx3-recycling tubules.

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.

The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane-localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C-terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post-ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane.

Coursework that is dependent on experiential learning, such as that offered in the laboratory-based sciences, can present barriers for a variety of students. These barriers can be magnified for students who experience disabilities that impact their access to the full depth of the course content and materials. Our unique perspective was formed primarily by supporting two blind students at two institution types across multiple biological science departments. In this paper, we explain the barriers we find are currently impacting blind and visually impaired (VI) students’ access to STEM as well as some tools to navigate creating more equitable and accessible spaces to support blind/VI students in STEM. More specifically, we provide general recommendations for working with blind/VI students using a student-centered approach to planning and daily interactions, followed by tips and tools for preparing accessible materials and a case study on how to use learning outcomes to modify course activities to be more useful for blind/VI students, and lastly we provide recommendations for important collaborations.