Production of Reagents and Optimization of Methods for Studying Calmodulin-Binding Proteins

Abstract

Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins from Dictyostelium discoideum likely necessitates the use of CaM from the same organism. In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities. The gene encoding D. discoideum CaM has previously been cloned allowing production of recombinant protein. The present study describes the expression of D. discoideum CaM in Escherichia coli and its straightforward and rapid purification. Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities. Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments. The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix. The effectiveness of this methods is illustrated by the detection of potentially novel D. discoideum CaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I.

The AP-1 Clathrin-adaptor Is Required for Lysosomal Enzymes Sorting and Biogenesis of the Contractile Vacuole Complex inDictyosteliumCells

“…In contrast, Rh50 and VatA did not colocalized to the CV network in apm1 Ϫ and apm1 Ϫ ϩ 1⌬Ct mutant cells ( Figure 10A). In addition, Calmodulin, another CV component (Ultricht and Soldati, 1999), also redistributed in endosomes in apm1 Ϫ cells, and the characteristic CV network was not detected in apm1 Ϫ mutant cells incubated with FM 4-64 (a fluorescent dye known to label the CV complex; our unpublished results). In addition, Calmodulin, another CV component (Ultricht and Soldati, 1999), also redistributed in endosomes in apm1 Ϫ cells, and the characteristic CV network was not detected in apm1 Ϫ mutant cells incubated with FM 4-64 (a fluorescent dye known to label the CV complex; our unpublished results).…”

Rab8a regulates the exocyst-mediated kiss-and-run discharge of theDictyosteliumcontractile vacuole

“…The following antibodies were used: a mouse monoclonal antibody against the A subunit of the V-ATPase complex (VatA; monoclonal antibody, 221-35-2; Neuhaus et al , 1998) and rabbit polyclonal antibodies against Dgap1 (Faix and Dittrich, 1996), Rhesus50 (Benghezal et al , 2001), and calmodulin (Ulbricht and Soldati, 1999). The antibodies against mRFP, Sec3, and Rab8a were obtained during this study by immunization of rabbits with purified mRFP protein, purified GST-Sec3 N-terminus (amino acids [aa] 1–188), and a synthetic Rab8a peptide (aa 172–186: NH2-C-DIKKRMIDTPNEQPQ-CONH2).…”

Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

“…Cells were not allowed to reach a density of more than 2ϫ10 6 cells/ml. Antibodies used were a rabbit antiserum to the Cterminal cytosolic tail of Rh50 (controls showed that pFL759 was not recognized by this antiserum) ), a rabbit antiserum to calmodulin (a kind gift from T. Soldati, University of Geneva, Switzerland) (Ulbricht and Soldati, 1999) and a mouse monoclonal antibody to CsA (mAb 41-71-21) (Bertholdt et al, 1985). Note that in our cell culture conditions (low density), endogenous CsA was not expressed and therefore this anti-CsA antibody only recognized CsA-Rh50 chimeras.…”