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Figure 5.
Fig. 5. The actin binding and dimerization functions of CAP
are mediated by separate domains. A, the last 27 amino acids of
CAP are essential for actin binding.  cap yeast
cells (SKN32) bearing plasmids expressing HA-CAP (YCpADH-HACAP)
and Myc-CAP1-498 (pADH-mycCAP 11) were
grown to log phase, prior to harvesting, and lysate preparation.
B, CAP1-498 forms homodimers. cap yeast
cells (SKN32) bearing plasmids expressing HA-CAP1-498
(pTADH-HACAP 11) and
Myc-CAP1-498 (pADH-mycCAP 11) were
grown to log phase, prior to harvesting and lysate preparation.
Immunoprecipitations were carried out, as described under
``Experimental Procedures,'' using either anti-HA ascites
(12CA5) or anti-Myc antisera (9E10). Specific protein-protein
interactions were blocked by the addition of either excess HA
(30 µg) or Myc peptide (30 µg) to
immunoprecipitation reactions (+peptide). Detection of tagged
CAP molecules or actin was accomplished using the appropriate
monoclonal antibodies (anti-actin (6 µg/ml), anti-HA
ascites fluid (1:5000), and anti-Myc (1:5000)). Lanes marked TCL
contained 75 µg of total cell lysate. Antigen detection
was performed by ECL chemiluminescent assay.
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