Figure 1.

Determination of the PMN rate by Western blots. As detailed in the text GFP is rather resistant against vacuolar proteolysis. The level of GFP generated by breakdown of the NV junction proteins GFP-Osh1 (A) or Nvj1-GFP (B and C) therefore corresponds to the PMN rate. Cells of the stationary growth phase were shifted to nitrogen-free SD(-N) medium, at the indicated times aliquots were taken, and crude extracts were prepared. The samples were then separated by SDS-PAGE and after electroblotting onto PVDF membranes were probed with antibodies against GFP and a secondary antibody coupled to peroxidase. Chemiluminescence was detected with a Fuji LAS3000. The absence of free GFP in cells lacking vacuolar proteinase A (pep4Δ) confirms the vacuolar origin of GFP. As a loading control the blots were reprobed with antibodies to cytosolic 3-phosphoglycerate kinase (PGK). (B) Nvj1-GFP is expressed under control of the copper-inducible CUP1 promotor. The expression level was checked in the presence of increasing copper concentrations. (C) Expression of CUP1::Nvj1-GFP was induced for 2 h with 5 μM Cu²⁺, and then the cells were shifted to starvation medium and treated as above.