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The objective of this study was to determine how active transporters (pumps), located at the plasma membrane of a cell, are regulated. We use two species of yeast (Schizosaccharomyces pombe and Saccharomyces cerevisiae) as model Eukaryotes to study how such pumps are endocytosed. In eukaryotes, endocytosis is initiated once membrane proteins are ubiquitinated. This ubiquitination tag allows the cell to properly select membrane transporters and place them into endocytic vesicles. Ubiquitination is determined by a ubiquitin ligase/adaptor complex; featuring a core ubiquitin ligase enzyme bound to a variety of arrestin family adaptors which determine which pumps are targeted. Previous studies in cerevisiae have suggested that a positively charged basic groove on the arrestin Art1 interacts with a negatively charged acidic patch binding site on the methionine pump Mup1. Charge inversion experiments provide key evidence for this model: adding positive charges to the Mup1 acidic patch completely blocks endocytosis, and compensating negative charges added to the Art1 basic groove restore it. The exact position of the charge inversion pairs is highly specific, suggesting lock and key binding. We set out to test whether this mechanism of binding also occurs in S. pombe for the pump/adaptor pair Cat1/Any1 (which are structurally similar and closely related to Mup1/Art1). Here we test the acidic patch/basic groove binding model by systematically mutating the cytoplasmic surface of the Cat1 pump. Course grain mutation mapping identified a 5 amino acid, negatively charged acidic patch that is indeed critical for endocytosis. We are currently testing the effect of introducing positive charges in this region, and introducing corresponding negative charges into the hypothesized Any1 basic groove.
The objective of this study was to determine how active transporters (pumps), located at the plasma membrane of a cell, are regulated. We use two species of yeast (Schizosaccharomyces pombe and Saccharomyces cerevisiae) as model Eukaryotes to study how such pumps are endocytosed. In eukaryotes, endocytosis is initiated once membrane proteins are ubiquitinated. This ubiquitination tag allows the cell to properly select membrane transporters and place them into endocytic vesicles. Ubiquitination is determined by a ubiquitin ligase/adaptor complex; featuring a core ubiquitin ligase enzyme bound to a variety of arrestin family adaptors which determine which pumps are targeted. Previous studies in cerevisiae have suggested that a positively charged basic groove on the arrestin Art1 interacts with a negatively charged acidic patch binding site on the methionine pump Mup1. Charge inversion experiments provide key evidence for this model: adding positive charges to the Mup1 acidic patch completely blocks endocytosis, and compensating negative charges added to the Art1 basic groove restore it. The exact position of the charge inversion pairs is highly specific, suggesting lock and key binding. We set out to test whether this mechanism of binding also occurs in S. pombe for the pump/adaptor pair Cat1/Any1 (which are structurally similar and closely related to Mup1/Art1). Here we test the acidic patch/basic groove binding model by systematically mutating the cytoplasmic surface of the Cat1 pump. Course grain mutation mapping identified a 5 amino acid, negatively charged acidic patch that is indeed critical for endocytosis. We are currently testing the effect of introducing positive charges in this region, and introducing corresponding negative charges into the hypothesized Any1 basic groove.
