The retromer and Snx4/41/42 pathways independently recycle Can1, and Ere1 functions in the retromer pathway. (A) Loss of both retromer and Snx41 function results in additive resistance to canavanine (top), whereas loss of multiple retromer subunits is not additive (bottom). (B) Loss of both Ere1 and Snx41 confers an additive canavanine resistance phenotype, but loss of both Ere1 and the retromer subunit Snx3 is not additive. (A, B) Serial dilutions of wild-type and various mutant cells (as indicated) were grown on synthetic media plates with or without 2 μg/ml canavanine for 2 d at 26°C. (C) Rates of Can1 degradation are increased in cells lacking Snx41, Snx3, or Ere1. Double deletion of ERE1 and SNX41 further increases the rate of Can1 turnover. Wild-type and mutant cells (as indicated) expressing FLAG-tagged Can1 were grown to mid-log phase and treated with cycloheximide to induce Can1 endocytosis. Cell lysates were prepared at the indicated time points and analyzed by immunoblotting to monitor Can1 degradation rates (top; t1/2 times are shown on the right). G6PDH was used as a protein loading control (bottom).
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