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First published online March 21, 2007
doi: 10.1242/10.1242/jcs.000158

Journal of Cell Science 120, 1225-1234 (2007)
Published by The Company of Biologists 2007
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Mechanism and biological role of profilin-Srv2/CAP interaction

Enni Bertling1, Omar Quintero-Monzon2, Pieta K. Mattila1, Bruce L. Goode2,* and Pekka Lappalainen1,*

1 Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland
2 Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, MA 02454, USA


Figure 1
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  		Fig. 1. Srv2 binds directly to profilin (KD=1.3 µM). (A) Profilin binding to GST-Srv2253-526 on glutathione agarose beads measured by supernatant depletion pull-down assays. Reactions contained 2 µM wild-type profilin (Pfy1) or mutant profilin with impaired binding to polyproline (Pfy1-14) and variable concentrations of Srv2 (0-20 µM). The amount of Pfy1 in the supernatant was examined by SDS gel electrophoresis. First lane, no beads; second lane, controlbeads; lanes 3-6, 3, 6, 12 and 20 µM Srv2 immobilized on beads. (B) Quantification of five independent pull-down assays comparing Pfy1 with Pfy1-14. Srv2 efficiently depleted Pfy1 from the supernatant, but failed to deplete Pfy1-14. Standard deviations are indicated with error bars. (C) Tryptophan fluorescence assay for determining the dissociation constant of the Srv2-profilin complex. Reactions contained 1 µM profilin. Pfy1 binds to Srv2253-373 with a KD of 1.3 µM, whereas Pfy1-14 shows no detectable affinity for Srv2253-373.

 

Figure 2
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  		Fig. 2. The P1 polyproline sequence is required for Srv2 binding to profilin. (A) Sequence alignment of the two mouse Srv2/CAP isoforms (CAP1 and CAP2) and yeast Srv2. Positions of the proline-to-alanine substitutions in Srv2-201, Srv2-202 and Srv2-203 mutants are indicated below the sequences. (B) Quantification of four independent supernatant depletion pull-down assays. Wild-type Srv2 and Srv2-203 bind profilin efficiently, whereas Srv2-201 and Srv2-202 fail to bind profilin in this assay. Standard deviations are indicated by error bars. (C) Tryptophan fluorescence assay of wild-type Srv2253-373 and mutant Srv2-201253-373. Wild-type Srv2253-373 but not Srv2-201253-373 induces a saturable increase in tryptophan fluorescence of 1 µM profilin.

 

Figure 3
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  		Fig. 3. Interactions of wild-type Srv2 and mutants Srv2-201, Srv2-202 and Srv2-203 with ADP-G-actin. An increase in the fluorescence of 0.2 µM NDB-labeled G-actin was measured under physiological salt conditions at pH 8.0. Dissociation constants were calculated from the binding curves. Srv2, Srv2-201 and Srv2-203 bound to actin monomers with high affinity (KD=0.066 µM, 0.056 µM and 0.074 µM, respectively), whereas Srv2-202 bound actin monomers with considerably lower affinity (KD=0.4 µM).

 

Figure 4
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  		Fig. 4. Actin and profilin do not interfere with each other's binding to Srv2. (A) The presence of profilin does not alter the affinity of Srv2 for G-actin. Addition of 1 µM Srv2 or Srv2-201 to 0.2 µM NBD-labeled ADP-G-actin resulted in a ~30% increase in the fluorescence. Addition of profilin (0-40 µM) did not significantly reduce the NBD fluorescence signal, suggesting that profilin does not affect the binding of Srv2 or Srv2-201 to ADP-G-actin. Standard deviations are indicated by error bars. (B) Actin-binding does not change Srv2 affinity for profilin. Supernatant depletion pull-down assays were carried out with reactions containing 2 µM profilin; the average of five independent assays is shown. Lane 1, profilin alone; lanes 2-6, 20 µM GST-Srv2 on beads; lanes 3-6, variable concentrations of ADP-G-actin (1, 2, 4, 10 µM). Addition of ADP-G-actin does not change the amount of profilin in the supernatant, demonstrating that actin monomers do not interfere with Srv2-profilin interaction. Results using Srv2-201 show that profilin does not bind indirectly to Srv2 through interaction with G-actin in this assay. Standard deviations are indicated by error bars.

 

Figure 5
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  		Fig. 5. Srv2 interacts with profilin in vivo. (A) Immunoprecipitation of Srv2 with anti-Srv2 antibody was carried out from wild-type SRV2, srv2-201 and srv2{Delta} yeast strains. The blot was probed with anti-profilin antibody and demonstrates specific co-immunoprecipitation of profilin with wild-type Srv2. By contrast, only very small amounts of profilin were detected in immunoprecipitates from srv2-201 and srv2{Delta} cells. The amounts of profilin in cell lysates before co-immunoprecipitation are shown as a control. (B) Srv2 localizes in patch-like manner in SRV2 and srv2-201 cells. The Srv2 dots occasionally co-localize with cortical actin patches.

 

Figure 6
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  		Fig. 6. The srv2-201 allele suppresses defects in cell growth and actin organization caused by pfy1-4. (A) Comparison of cell growth defects for progeny from a cross between pfy1-4 and srv2-201 strains. Diploids from this cross were sporulated, tetrads were dissected. Lethality of spores was scored on the tetrad plates. Viable haploid strains from these plates were re-plated on YPD and scored for cell growth at 25 and 37°C. n, number of haploid progeny with the indicated genotype analyzed. (B) Transformation assay showing that srv2-201 suppresses the growth defects of pfy1-4. Double mutant pfy1-4, srv2-201 cells were transformed with a low copy URA-marked empty vector (pRS316) or pSRV2 (pBG334). Serial dilutions of transformed cells were plated on Ura- selective medium and grown at 25, 30, 34 and 37°C. As a control, double mutant aip1, srv2-201 cells were transformed with same vectors. (C) Cells from A above were grown to log phase, chemically fixed and stained with Alexa Fluor 488-phalloidin to label F-actin structures. (D) Quantitative comparison of actin patch polarization defects in pfy-4 and pfy1-4 srv2-201 cells. Medium-budded cells (n>200, each strain) were scored for actin patch distribution in the bud. Actin patches were classified as being polarized, unpolarized or intermediate polarized.

 




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