Biological membranes organize their proteins and lipids into nano- and microscale patterns. In the yeast plasma membrane (PM), constituents segregate into a large number of distinct domains. However, whether and how this intricate patchwork contributes to biological functions at the PM is still poorly understood. Here, we reveal an elaborate interplay between PM compartmentalization, physiological function, and endocytic turnover. Using the methionine permease Mup1 as model system, we demonstrate that this transporter segregates into PM clusters. Clustering requires sphingolipids, the tetraspanner protein Nce102, and signaling through TORC2. Importantly, we show that during substrate transport, a simple conformational change in Mup1 mediates rapid relocation into a unique disperse network at the PM Clustered Mup1 is protected from turnover, whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its own turnover. Our findings suggest that lateral compartmentalization provides an important regulatory link between function and turnover of PM proteins.

1 Biological membranes organize their proteins and lipids into nano-and microscale 2 patterns. In the yeast plasma membrane (PM) constituents segregate into a large number 3 of distinct domains. However, if and how this intricate patchwork contributes to 4 biological functions at the PM is still poorly understood. Here, we reveal an elaborate 5 interplay between PM compartmentalization, biochemical function and endocytic 6 turnover. Using the methionine permease Mup1 as model system we demonstrate that 7 this transporter segregates into PM clusters. Clustering requires sphingolipids, the 8 tetraspanner Nce102 and TORC2 signaling. Importantly, we show that during substrate 9 transport, a simple conformational change in Mup1 mediates rapid relocation into a 10 unique disperse network at the PM. Clustered Mup1 is protected from turnover, 11whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its 12 own turnover. Our findings suggest that lateral compartmentalization provides an 13 important regulatory link between function and turnover of PM proteins. 14 15

Topography is a critical feature driving formation and dynamics of protein and lipid domains within biological membranes. The yeast plasma membrane (PM) has provided a powerful model system to study lateral domain formation, including characteristic BAR domain-induced PM furrows. Currently, it is not clear how the components involved in the establishment of these furrows cooperate to precisely regulate local PM topography. Here we report opposing functions for the Sur7 and Nce102 families of tetraspanner proteins in modulating membrane curvature and domain topography. Using STED nanoscopy and freeze-fracture EM we found that Sur7 tetraspanners form multimeric strands at the upper edges of PM furrows, which counteract the forces exerted by BAR domain proteins and prevent membrane tubulation. In contrast, Nce102 tetraspanners are located basal to the Sur7 proteins and promote BAR domain-induced curvature. The segregation of the two tetraspanner-based nanodomains is further supported by differential distribution of ergosterol to the upper edge of furrows and PIP2 lipids at the furrow base. These findings suggest a general role of tetraspanner proteins in sculpting local membrane domains.