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Essential functions of Sds22p in chromosome stability and nuclear localization of PP1

Mark W. Peggie1, Sarah H. MacKelvie1,*, Andrew Bloecher2,{ddagger}, Elena V. Knatko1, Kelly Tatchell2 and Michael J. R. Stark1,§

1 Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK
2 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
* Present address: Scottish Enterprise Tayside, 45 North Lindsay Street, Dundee, DD1 1HT, UK
{ddagger} Present address: Fred Hutchinson Cancer Research Center, Division of Basic Research, 1100 Fairview Avenue North, Seattle, WA 98109, USA



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  		Fig. 1. Nuclear localization of Sds22p. Sds22p-myc3 and HA-Glc7p were localized by indirect immunofluorescence microscopy using strain SAY342 and data presented for three representative cells. A,F,K, DAPI fluorescence; B,G,L, HA-Glc7p; C,H,M, Sds22p-myc3; D,I,N, merged HA-Glc7p/Sds22p-myc3; E,J,O, merged DAPI/HA-Glc7p/Sds22p-myc3. Bar, 2 µm.

 


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  		Fig. 2. The Sds22p-Glc7p complex. (A) Proteins bound by protein A-tagged Sds22p (Sds22p-PrA) were recovered from extract of strain SAY1230 after affinity isolation on IgG-Sepharose followed by elution of Sds22p protein complexes with TEV protease, which cleaves between Sds22p and the protein A tag. Proteins present in the eluate when control extracts in which the protein A tag alone (PrA) was expressed are also shown. Comparison of the two lanes shows only two major bands specific to the Sds22p-PrA eluate that were identified by mass spectrometry as Glc7p and Sds22p. (B) Proteins co-purifying with protein A tagged Glc7p (PrA-Glc7p) were prepared similarly but from strain LKY150 and the two major bands indicated were identified as in A. In addition to Sds22p, several other higher molecular weight proteins ( ->)were present. (C) Protein extract (30 µg, lanes 1,3; or 120 µg, lanes 2,4) from a strain (LKY168) expressing HA-Glc7p and Sds22p-PrA was immunodepleted of HA-Glc7p using anti-HA antibodies and the amount of HA-Glc7p or Sds22p-PrA remaining after immunodepletion (supernatant) compared with the initial level (total) by western blot analysis. (D) Control experiment using a strain with untagged Glc7p (SAY1228), confirming that Sds22p-PrA is not depleted by the anti-HA antibodies used in (C).

 


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  		Fig. 3. Reciprocal genetic interactions between SDS22 and GLC7. Tenfold serial dilutions of wild-type, sds22-6, glc7-5 and glc7-12 strains transformed with empty vector (YEplac195) or high-copy YEplac195 constructs carrying SDS22 or GLC7 were plated on YPD agar at different growth temperatures. Partial suppression of sds22-6 by high-copy GLC7 and of both glc7 alleles by high-copy SDS22 can be seen by growth of the appropriate strains at normally restrictive temperatures.

 


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  		Fig. 4. Release of sds22 Ts– mutants from alpha-factor arrest. Wild-type, sds22-5 and sds22-6 cells were synchronized in G1 using alpha-factor and released at either 26°C or 37°C. Cell density and % budded cells were monitored and the DNA content measured by FACS analysis. At 37° C the mutant cells budded and replicated their DNA, but proliferation ceased after an approximately twofold increase in cell density. {circ}, wild-type (SAY306); {square}, sds22-5 (SAY302); {blacklozenge}, sds22-6 (SAY304).

 


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  		Fig. 5. Stability of Sds22p and Glc7p in the sds22-6 mutant. Strains solely dependent on myc epitope-tagged wild-type (Sds22p-myc3: SAY326) or mutant (Sds22-6p-myc3: SAY284) Sds22p or wild-type (MPY1165) and sds22-6 (MPY1171) strains expressing HA epitope-tagged Glc7p (HA-Glc7p) were shifted to 37°C and the stability of either protein monitored by western blot analysis over 6 hours. The level of calmodulin (Cmd1p) is shown to demonstrate equivalent loading of each lane and the growth curve indicates cell density at each time point.

 


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  		Fig. 6. Effect of sds22-6 on the Sds22p-Glc7p complex. Strains dependent on untagged Sds22p (SAY350, SAY352; lanes 1,2) or myc epitope-tagged wild-type (Sds22p-myc3: SAY342, SAY344; lanes 3,4) or mutant (Sds22-6p-myc3: SAY338, SAY340; lanes 5,6) Sds22p were transformed with plasmids encoding either HA-Glc7p (lanes 1,3,5) or the equivalent HA-tagged glc7-12 allele (lanes 2,4,6). Cells were grown at 26°C and myc-tagged Sds22p recovered from extracts by immunoprecipitation after incubation at either 4°C or 30°C. The Sds22p recovered and the amount of co-precipitable Glc7p were visualized by western blot analysis with either anti-myc (left panel) or anti-HA (right panel) antibodies.

 


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  		Fig. 7. Suppression of the ipl1-2 temperature-sensitive phenotype by sds22-6. Tenfold serial dilutions of wild-type (EG1085-10A), ipl1-2 (EG1085-4B) sds22-6 (EG1085-12A) and ipl1-2 sds22-6 (EG1085-12C) were plated onto YPD agar and incubated for 2 days at the indicated growth temperatures.

 


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  		Fig. 8. Glc7p localization is affected in the sds22-6 mutant. Diploid SDS22/SDS22 (KT2070), sds22-6/sds22-6 (KT2066) and SDS22/sds22-6 (KT2067) cells expressing GFP-Glc7p were grown at 22°C and then shifted to 37°C. The effect of the temperature shift on wild-type and mutant cells is presented for a representative field, showing DIC and GFP fluorescence images for each time point.

 

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© The Company of Biologists Ltd 2002