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Relationship between the function and the location of G1 cyclins in S. cerevisiae

Nicholas P. Edgington and Bruce Futcher*

Department of Molecular Genetics and Microbiology, Life Sciences Bldg., SUNY at Stony Brook, Stony Brook, NY 11794-5222, USA



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  		Fig. 1. Construction of the forced localization cassettes. (a) Cassettes containing two NLS or NES localization motifs separated by a T7 epitope tag were added to the C-terminus of Cln2 and Cln3. (b) Active and inactive versions of both a SV40 nuclear localization signal (NLS) and a PKI nuclear export signal (NES) were created. Inactive versions contain point mutations previously shown to prevent transport activity in S. cerevisiae (Nelson and Silver, 1989) (Murphy and Wente, 1996). (c) The localization cassettes direct GFP to the expected locations. Forced localization cassettes fused to GFP under control of the GAL1,10 promoter were grown to mid-log growth phase. GFP expression was induced by the addition of 0.1% galactose for 1-2 hours, and live cells were subsequently stained, observed and digitally captured as described in Materials and Methods. DIC, differential interference contrast; Hoechst represents DNA staining; –A represents the active/functional transport sequences; –I represents the inactive/nonfunctional transport sequences.

 


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  		Fig. 2. Cln2 is located in both the nucleus and the cytoplasm. Strain N-5 containing an empty plasmid ({Delta}) or the plasmid pSL46(GAL::CLN2-3xHA) (HA) was grown at 30°C in selective media containing 2% raffinose to 5x106 cells/ml. The culture was divided in two, and galactose was added to one of them (1% final). All four cultures (+/– CLN2-3xHA, and +/– galactose induction) were grown for an additional hour then collected and fractionated. (a) Cytoplasmic and nuclear fractions from induced cln2{Delta} or GAL-CLN2-3xHA cells were analyzed using SDS/PAGE and blotted onto a membrane. 20 µg of protein from the cytoplasmic fraction, and 20 µg of protein from the nuclear fraction, were loaded. The western blot was first probed with anti-HA 12CA5 monoclonal antibody to detect Cln2, and subsequently reprobed with an anti-ADH polyclonal antibody as a cytoplasmic control, and a monoclonal antibody that recognizes the nuclear protein Nop1 (a kind gift from J. Aris, University of Florida, FL). (b) Cytoplasmic and nuclear fractions from uninduced (–) or induced (+) cells bearing GAL-CLN2-3xHA were analyzed using SDS-PAGE and blotted onto a membrane. 7 µg of protein was loaded. The western blot was probed with an anti-Sic1 polyclonal antibody (kindly provided by J. Donovan, SUNY at Stony Brook, NY). The top panel shows a high molecular weight nonspecific band as an internal loading control. (c) Plasmid pSL122(GAL::CLN24T3S-3xHA) was introduced to strain N-5, and treated as in (a). Equal amounts of protein were loaded in each lane. The blot was probed with anti-HA (12CA5) antibody to detect Cln2-3xHA, and subsequently reprobed with an anti-ADH polyclonal antibody, and anti-NOP1 monoclonal antibody.

 


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  		Fig. 3. Cln2 localizes to the cytoplasm and to the nucleus, and the forced localization cassettes can shift Cln2 to specific compartments. (a) GFP-CLN2-18xMYC (plasmid pMGH101) was introduced to strain N-104(#31) (cln1 cln2 GAL-CLN3 cdc34-2), which also contains a centromeric plasmid containing CLN3 under its own promoter (pHW415). Cells were grown under selective conditions (–met 2% glucose) at 25°C to early log phase, and then shifted to 37°C for 1-3 hours. Live cells were stained with Hoechst 333642, mounted under coverslips in mounting media, and images were captured digitally. The cell on the right rotated slightly between imaging. (b) Plasmids (pMGH10X series) containing GFP-CLN2 fused to the forced localization cassettes at the Cln2 C-terminus were introduced to strain N-104(#31) and analyzed as described in (a).

 


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  		Fig. 4. Cln2 performs important roles in both the nucleus and the cytoplasm. (a) An empty control vector (pRS315), a low-copy plasmid containing CLN2 with no tag (CLN2/pCB1314) and plasmids containing CLN2 with C-terminal forced localization cassettes under control of the CLN2 promoter (pNE10X series) were transformed into strain N-138 (cln1 cln2 cln3 GAL-CLN1). Cells were serially diluted (see Materials and Methods) onto selective plates containing 2% galactose (GAL) or glucose (GLC), and grown for 2 days at 30°C. Within a single dilution series, the number of cells in each spot differs by tenfold from its neighbor (b) An empty control vector (pRS315), a low copy plasmid containing CLN2 with no tag (CLN2/ pNE113) and plasmids containing CLN2 with C-terminal forced localization cassettes under control of the methionine-repressible MET3 promoter (pM10X series) were transformed into strain YHW23 (swi4 swi6 GAL-SWI4). Strains were serially diluted as in (a) on plates containing 2 mM methionine or lacking methionine (–MET) and containing 2% galactose or glucose. (c) The same plasmids were used as in (b), but were transformed into strain N-162 (swi4 swi6 GAL-SWI4 sic1), and cells were grown for 5 days at 30°C.

 


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  		Fig. 5. Cln2 shares nuclear functions with the S-phase cyclins Clb5 and Clb6, and with the kinesin motor protein Kar3, and shares cytoplasmic functions with the cyclin pair Pcl1 and Pcl2, and with GTPase-activating protein Bud2. (a) The same plasmids as in Fig. 4, the pNE10X series and the pM10X series, were introduced into strain N140 (cln1 cln2 clb5 clb6 GAL-CLN1), and processed as described in Fig. 4. The left panel are serial dilutions that were incubated at 30°C for 2 days, and the right panel were incubated at 34°C for 3 days. (b) Plasmids containing CLN2 with C-terminal forced localization cassettes under control of the methionine-repressible MET3 promoter (pM10X series) were introduced into strain N182 (cln1 cln2 kar3 GAL-CLN1). (c) The same plasmids as in (a) were introduced to the strain N80 (cln1 cln2 pcl1 pcl2 GAL-CLN1), and processed as described in Fig. 4. (d) An empty control vector (pRS314), a low-copy plasmid containing CLN2 with no tag (CLN2/p314M::C2) and plasmids containing CLN2 with C-terminal forced localization cassettes under control of the methionine-repressible MET3 promoter (pNE12X series) were transformed into the strain N163 (cln1 cln2 bud2 GAL-CLN1) and processed as previously described.

 


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  		Fig. 6. Cln2 may need to shuttle between nucleus and cytoplasm. Strain N-225 (swi4 swi6 GAL-SWI4) was doubly transformed so as to contain two plasmids simultaneously, one of the pNE12X series, and one of the pM10X series. Strains were serially diluted and plated as described in Fig. 4, except that growth was for 2 days or 4 days (panels on right).

 


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  		Fig. 7. Cln3 must be in the nucleus to function. (a) Cell volumes are shown in femtoliters. Various versions of CLN3 (plasmids pBF580, pBF580HA, pNE110, pNE111, pBF{Delta}528HA, pBF{Delta}528NLS-A, and pBF{Delta}528NLS-I) were integrated in single copy at the CLN3 locus in the parental strain ML201:699(cln3::LEU2). These strains were grown in YEPD, and cell volumes were measured with a Coulter Channelyzer (Yaglom et al., 1995). The mode volume is reported. (b) The same plasmids containing alleles of CLN3 used in (a) were integrated in single copy into the strain FC-1254-14D (cln1 cln2 cln3 GAL-CLN1). Cells were grown overnight in YEP 2% galactose, and tenfold serial dilutions were spotted onto YEP plates containing either 2% galactose or 2% glucose. Plates were photographed after 2 days at 30°C. (c) CLN3 C-terminal amino acids encompassing either the whole (pMG61, 24 aa) or a portion (pMG31, 16 aa; pMG40, 5 aa) of the putative bipartite NLS were fused to GFP under control of the MET3 promoter. Residues in bold show the NLS. Methionine residues, in parentheses, were introduced to facilitate cloning. (d) Plasmids containing the GFP fusions described in (c) were introduced to strain N-5. Cells were grown to log-phase in medium containing 2 mM methionine and 2% glucose, induced for 3 hours by resuspension in medium lacking methionine, and images were collected.

 

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