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Casein kinase I controls a late step in the endocytic trafficking of yeast uracil permease

Christelle Marchal^*, Sophie Dupré^* and Daniele Urban-Grimal

Institut Jacques Monod, CNRS-UMRC9922, Université Paris 6 and Paris 7-Denis Diderot, 2 place Jussieu, 75251-Paris-cedex 05, France
^* These authors contributed equally to this work

View larger version (19K): [in a new window]   Fig. 1. The GFP-tagged uracil permease is correctly delivered to the plasma membrane. Wild-type cells transformed with p197gF or p195gF-GFP (A) or pFl38gF-GFP (B) and grown on lactate as a carbon source were induced by growth for two hours on galactose medium. (A) Uracil permease activity was followed at various times after addition of galactose. (O) and () corresponded to cells transformed with p197gF and p195gF-GFP respectively. (B) Cell fluorescence was followed at various times after addition of galactose. Fluorescence staining was only observed in budding cells. Cells were viewed with a FITC filter set.

View larger version (29K): [in a new window]   Fig. 2. Internalization and degradation of uracil permease are impaired in yckts cells. Wild-type, apm3-, yckts and yckts apm3- cells transformed with pFL38gF-GFP were grown to the exponential growth phase at 24°C with galactose as a carbon source. Cells were incubated at 37°C for 20 minutes. Adding glucose and incubating for 10 minutes at the same restrictive temperature stopped the synthesis of Fur4p-GFP. Cycloheximide (100 µg/ml) was then added. (A) Uracil uptake (permease activity) was measured at 37°C at various times after the addition of cycloheximide. The results are expressed as a percentage of the initial activity. (B) Protein extracts were prepared at the times indicated after the addition of cycloheximide. Aliquots were analyzed for uracil permease by western immunoblotting using anti-GFP antibody. *, blots were reprobed with anti-Pgk antibody to provide loading controls. I, mostly permease conjugated with ubiquitin. Open, gray and black circles corresponded to different levels of phosphorylation of the permease with the faster migrating bands corresponding to the lowest level of phosphorylation of the permease.

View larger version (81K): [in a new window]   Fig. 3. Nomarski optics and distribution of Fur4p-GFP fluorescence in wild-type (WT), apm3, yckts and yckts apm3- mutant cells. Wild-type, apm3, yckts and yckts apm3- cells transformed with pFL38gF-GFP were grown to the exponential growth phase at 24°C in galactose medium. Cells were incubated at 37°C for 20 minutes. The synthesis of Fur4p-GFP was stopped by adding glucose and incubating for 10 minutes at the same restrictive temperature. Cells were observed at various times after the addition of cycloheximide at 37°C. Cells were viewed by Normarski optics or with a FITC filter set.

View larger version (22K): [in a new window]   Fig. 4. Fur4p-GFP is retained in the class E compartment in vps27ts mutant cells. Parental and vps27ts mutant cells transformed with pFL38gF-GFP were grown to exponential growth phase at 24°C in galactose medium. Cells were transferred to 37°C for two hours. The synthesis of Fur4p-GFP was stopped by adding glucose and incubating for 10 minutes at the same restrictive temperature. Cycloheximide (100 µg/ml) was then added. (A) Uracil uptake (permease activity) was measured at 37°C at various times after the addition of cycloheximide. The results are expressed as a percentage of the initial activity. (B) Nomarski optics and distribution of Fur4p-GFP fluorescence in parental and vps27ts mutant cells. Cells were observed two hours after adding cycloheximide at 37°C. Cells were viewed by Normarski optics or with a FITC filter set.

View larger version (26K): [in a new window]   Fig. 5. Fur4p-GFP partially colocalizes with Pep12p in yckts cells. yckts cells transformed with pFL38gF-GFP were grown to exponential growth phase at 24°C in galactose medium. Cells were incubated at 37°C for 20 minutes. The synthesis of Fur4p-GFP was stopped by adding glucose and incubating for 10 minutes at the same restrictive temperature. Cells were observed 90 minutes after the addition of cycloheximide at 37°C. Cells were fixed and processed for immunofluorescence using the anti-Pep12p antibody followed by rhodamine-labeled goat anti-mouse IgG. GFP fluorescence was visualized with a FITC filter and Pep12p distribution was observed with a rhodamine filter. Regions of colocalization are shown in yellow on the overlay.

View larger version (48K): [in a new window]   Fig. 6. yckts cells show wild-type processing of the soluble vacuolar protein CPY and wild-type localization of V-ATPase. (A) Processing of the soluble vacuolar protein CPY. Wild-type (WT), apm3, yckts and yckts apm3- cells were grown to exponential growth phase at 24°C in glucose medium, then incubated at 37°C for 30 minutes. Cells were pulse-labeled for 5 minutes with [35S]-translabel, chased for the times indicated (min) and subjected to immunoprecipitation with CPY antiserum. The immunoprecipitates were analyzed by electrophoresis in 10% polyacrylamide-SDS gels, which were then subjected to fluorography. p1, ER form; p2, Golgi form; m, mature vacuolar form. (B) Immunolocalization of the vacuolar H+ATPase subunit Vat2p. Cells were grown to exponential growth phase at 24°C in galactose medium, then incubated at 37°C for 90 minutes with cycloheximide as described in Fig. 2. Cells were prepared for immunofluorescence with the anti-Vma2p/Vat2p antibody. Cells were visualized by Normarski optics or with a FITC filter set.

View larger version (32K): [in a new window]   Fig. 7. yckts cells show wild-type processing of the vacuolar protein ALP. Wild-type (WT), apm3, yckts and yckts apm3- cells were grown to exponential growth phase at 24°C, then washed and resuspended at the same density in fresh medium depleted of inorganic phosphate for three hours at 37°C. Protein extracts prepared from wild-type and mutant cells were analyzed by SDS-PAGE and western immunoblotting for ALP. We used as a control strains sec7, defective in intra-Golgi trafficking at the non-permissive temperature, and pep4, defective in vacuolar protease processing. Pep4p-dependent cleavage of pro-ALP (pro) results in the formation of mature ALP (m) and a small amount of the aberrant form of ALP (*). The mature form of ALP is formed in sec7 mutants during growth at the permissive temperature.

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