Figure 3.

Relationship between Atg18 and components of the retromer complex (A) Model of the retromer complex containing Vps35, Vps26 and Vps29 together with the SNX-BAR proteins Vps5 and Vps17. (B) Co-precipitation of Vps35-6xHA with GFP-Atg18 was increased in the absence of either Vps5 or Vps17. Vps35-6xHA was chromosomally expressed, while GFP-Atg18 was under the control of a MET17 promotor. (C) The amount of Vps35-6xHA bound to GFP-Atg18 is decreased in a vps26∆ strain and no binding could be detected in a vps29∆ strain. (D) Quantification of at least three independent co-immunoprecipitation experiments. The ratio of HA signal to GFP signal in the bound fraction was normalized to the wildtype. Statistical relevance was determined using an unpaired two-tailed t-test, error bars indicate SEM, asterisks indicate p-values. (E,F) Vps26 co-precipitates with Atg18 in the presence of Vps35, while Vps29 does not. Either Vps26 or Vps29 were chromosomally tagged with 6xHA and expressed together with GFP-Atg18 under a MET17 promotor control. (G,H) Vps5 competes with Atg18 for binding to Vps35 during nitrogen starvation. Vps35-6xHA was expressed chromosomally together with either GFP-Atg18 (G) or Vps5-GFP (H) under the control of a MET17 promotor. Co-IPs were performed before and after 2 h of nitrogen starvation. Either Vps5 (G) or Atg18 (H) were deleted (-) or constitutively overexpressed under the control of a GPD1 promotor (+++). (I,J) Quantification of at least three independent co-immunoprecipitation experiments. The ratio of HA signal to GFP signal in the bound fraction was normalized to the 0 h sample of the wildtype. Statistical relevance was determined using an unpaired two-tailed t-test, error bars indicate SEM, asterisks indicate p-values.