Crude epidermal extracts were obtained using the procedure described above and the same extraction buffer

Crude epidermal extracts were obtained using the procedure described above and the same extraction buffer. dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the Purmorphamine intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids. import assays (19,C21). However, while analyzing its subcellular location in leaf cells, we formerly observed that this protein was not exclusively targeted to plastids (19) but was also partly localized at the periphery of plant cells and in some locally concentrated dots in the cytosol. Thus, in cells from leaves, ceQORH shows a complex subcellular location pattern: in the plastid envelope (following import Rabbit Polyclonal to RHO into plastids) and outside plastids (implying that plastid import did not occur). Here, we show that the calmodulin isoform CaM57 is Purmorphamine a key player for this dual location, thus providing a so-far-unanticipated role for the Purmorphamine intriguing CaM-binding properties of hundreds of plastid proteins (22), despite the fact that no CaM or CaM-like proteins were identified in plastids. Results The plant ceQORH protein interacts with calmodulin In the present study, we provide several lines of evidence demonstrating the specific CaM-binding property of ceQORH. First, the natural plant ceQORH was enriched in the fraction eluted from a calmodulin-affinity resin when compared with its level in crude cell extracts (Fig. 1with those of the endogenous protein ecQOR (as a negative control), the closest bacterial homolog of plant ceQORH (Fig. 1ceQORH from crude plant cell extracts. Purification was performed on a CaM affinity resin (Stratagene). ceQORH and K12 QOR (ecQOR) proteins in SDS-PAGE analysis of crude bacterial extracts containing ceQORH or ecQOR proteins. ceQORH and ecQOR produced in bacteria (see ceQORH protein interacts with the CaM affinity resin (and is thus eluted from the column), whereas this is not the case for the recombinant ecQOR protein. The CaM-binding domain is located in the C terminus of ceQORH To establish the location of the CaM-binding domain of ceQORH, we first performed successive deletions of the ceQORH protein (Fig. 2(Fig. 2and and Fig. S2) within the helix or adjacent to this helix according to the recently established 3D structure of ceQORH (14). Three of these mutants could be isolated in which mutagenesis of positively charged and hydrophobic residues abolishes the interaction of ceQORH with CaM (Fig. 3and are crude bacterial extracts containing recombinant ceQORH fusions as described for are crude bacterial extracts containing recombinant ceQORH fusions as described in K12 used as a negative control; used as a positive control; ceQORH mutant 1; ceQORH mutant 2; ceQORH mutant 3. Note that, in Mut2, mutagenesis of only three Purmorphamine residues is sufficient to abolish interaction of ceQORH with CaM. CaM binding is neither essential for ceQORH targeting to chloroplasts nor required for the specific location of ceQORH to the plastid envelope To determine whether the CaM-binding properties of ceQORH are responsible for the targeting of this protein to the chloroplast envelope, we established stable transformants expressing one truncated form (lacking its C terminus, construct 5 in Fig. 2construct 11 in Fig. 3and 16 kDa) CaM isoforms and tens of CaM-like proteins (25, 26). Having previously noted that ceQORH was mainly present at the periphery of plant cells in epidermal tissues (19), we decided to assess the abundance of CaM isoforms in epidermal tissue compared with crude leaf extracts. As seen in Fig. 5 20 kDa) than classical (shorter) CaM isoforms (16 kDa). This high-molecular-mass signal was also enriched in the membrane fraction of epidermal cells (Fig. 51/1000 of total epidermal proteins) compared with crude leaf extract (1 ng of CaM in 20 g of crude leaf extract, 1/20,000 of total leaf proteins) (Fig. 5CaM1, AT5G37780) (Fig. 5similar to CaM53 from petunia). Open.