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doi: 10.1242/10.1242/jcs.00203

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Plasma membrane localization of the Yck2p yeast casein kinase 1 isoform requires the C-terminal extension and secretory pathway function

Praveen Babu, Joshua D. Bryan, Heather R. Panek^*, Solomon L. Jordan, Brynn M. Forbrich, Shannon C. Kelley, Richard T. Colvin and Lucy C. Robinson

Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
^* Present address: SUNY-Buffalo, Department of Biochemistry, 140 Farber Hall, Buffalo, NY 14214, USA

View larger version (88K): [in a new window]   Fig. 1. GFP-Yck2p induction results in accumulation of intracellular punctate fluorescence before localization to the plasma membrane. (A) Induction of GFP-Yck2p was monitored by immunoblot analysis (Panek et al., 1997) using an affinity-purified antiserum against GFP. Detection in extracts from glucose-grown cells is shown in the left panels, for pJB1 (pGal:GFP:YCK2; lane 1) in wild-type strain LRB939, for GFP-Yck2p expressed from a chromosomal copy under the control of the YCK2 promoter in strain LRB913 (lane 2), and from a high copy (2µ) plasmid (pL2.35; lane 3) in LRB939. For the right panels, cells were grown at 30°C in synthetic medium containing 2% raffinose (derepressing conditions) and galactose was added to 2% at the 0 minute time point. Extracts were prepared from culture aliquots taken at the time points indicated. Antiserum against phosphoglycerate kinase (PGK) was used to reprobe the blots for loading control. (B) DIC and fluorescence images of living cells treated as in B were captured at the indicated time points. Bar, 2 µm.

View larger version (74K): [in a new window]   Fig. 2. GFP-Yck2p fails to reach the plasma membrane in secretory pathway-defective strains. Cells of strains LRB936 (sec18), LRB937 (sec23-1), LRB933 (sec14-3), LRB932 (sec4-2), LRB934 (sec9-4) (A) and PBY052 (sec23-1 end4-1) (B) carrying pJB1 (pGAL1:GFP:YCK2) were grown to log phase at 24°C in synthetic media containing 2% raffinose as a derepressing carbon source. Galactose was added to 2% and cultures were split, with half of each culture incubated at permissive (24°C) and restrictive (37°C) temperatures. Fluorescence images of live cells were captured after 90 minutes incubation at the indicated temperature. Bars, 2 µm.

View larger version (30K): [in a new window]   Fig. 3. GFP-Yck2p deletion mutants. (A) Illustration of full length GFP-Yck2 fusion protein, C-terminal substitution mutants (Cys545,546Ser; CIIS), C-terminal truncation mutant, C-terminal deletion mutant with intact -Cys-Cys motif, catalytic domain deletion mutant, and consecutive smaller C-terminal deletions. GFP portions are shown in green and Yck2p portions in gray. Plasma membrane localization (+) or mislocalization (-) is indicated to the right of each fusion protein. Color coding for smaller deletions matches that in panel B. (B) Yck2p C-terminal amino acid sequence with deleted regions indicated as follows: yck2374-390p in red; yck2391-412p in yellow, yck2406-409p with underscore; yck2413-444p in green; yck2445-470p in dark blue; yck2471-498p in violet; yck2499-518p in light blue; yck2519-527p in orange; yck2528-540p in turquoise.

View larger version (77K): [in a new window]   Fig. 4. Localization of Yck2p C-terminal variants in sec mutants at permissive and restrictive temperatures. Cultures of strains LRB936 (sec18) and LRB934 (sec9) carrying pGAL1 plasmids expressing the indicated Yck2p variants were grown and treated as described for Fig. 2, except that the induction period was 120 minutes before images were captured. Bar, 2 µm.

View larger version (43K): [in a new window]   Fig. 5. Complementation analysis of Yck2p C-terminal variants. (A) Galactose-inducible plasmids were introduced into yckts strain LRB951 and cultures were grown in selective medium containing 2% raffinose as the carbon source, and then plated onto medium containing 2% galactose. (B) Plasmids encoding the indicated Yck2p variants were introduced into the yckts strain LRB951. Transformants were grown to mid-log phase inmedia selective for plasmid retention and 5 µl drops of each culture and of 1/10 diluted cultures were plated onto selective agar media at the indicated temperature. Plates were incubated for 24 (glucose) or 48 (galactose) hours at 24°C or at 37°C before photographing.

View larger version (70K): [in a new window]   Fig. 6. A Yck2p variant carrying an FTase signal instead of the -Cys-Cys signal does not require Akr1p for membrane association. Plasmids pJB1 (pGal:GFP:YCK2) and pL251 (pGal:GFP:YCK2-CIIS) were introduced into strains LRB906 (wild-type) and LZY103 (akr1/akr1). Transformants were grown to mid-log phase at 30°C in selective medium with 2% raffinose as the carbon source, then galactose was added to 2% and cultures were incubated for 2 hours before fluorescence images were captured. Bar, 2 µm.

View larger version (110K): [in a new window]   Fig. 7. GFP-Yck2p C-terminal deletion mutants are mislocalized. Galactose-inducible plasmids encoding the indicated fusion proteins were introduced into wild-type strain LRB906 by transformation. Cells were grown on solid selective medium containing galactose as a carbon source at 30°C overnight, and were suspended in selective medium for capture of fluorescence images. Bar, 2 µm.

View larger version (34K): [in a new window]   Fig. 8. GFP-yck2519-527p fails to accumulate to normal levels. Immunoblot showing steady-state GFP-Yck2p, GFP-yck2-C545,546Sp and GFP-yck2519-527p levels in wild-type (LRB906) cells transformed with low-copy plasmids encoding the indicated fusion proteins. Protein samples were separated and transferred to nitrocellulose as described in Materials and Methods. Membranes were probed independently with GFP and PGK antibodies.

View larger version (70K): [in a new window]   Fig. 9. A GFP-Yck2/Yck1p chimera is modified and targeted to the plasma membrane similarly to GFP-Yck2p. (A) Sequence alignment of Yck2p and Yck1p C-terminal sequences. The sequences swapped in the Yck2/Yck1p chimera are shown. (B,C) Plasmid pL240, expressing GFP-Yck2/Yck1p upon galactose induction, was introduced into wild-type strain LRB906 and akr1/akr1 strain LZY103 (B) and into sec9, sec14 and sec23 strains (C). (B) Cells were grown to log phase in selective medium containing 2% raffinose as the carbon source, then galactose was added to 2% and cells were incubated at 30°C for 120 minutes before photographing. (C) Cells were grown and treated as described for Fig. 2 except that the induction period was 120 minutes. Bar, 2 µm.

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