Figure 5

Binding affinity of various truncated MFP polypeptides to MTs. (A) Polypeptide map of wild type MFP and four deletion polypeptides of MFP (C1, C2, N1, N2) used for the expression of recombinant proteins in E. coli and GFP chimeras in onion epidermal cells. Arrow indicates the location of the putative KXGS MT-binding domain. This putative domain is disrupted between the leucine and valine residues (see Results) in the truncated polypeptides C2 and N1. (B) The recombinant proteins were used in MT co-sedimentation assays to generate MT-binding curves. Increasing concentrations of each truncated protein were used (0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 7.5, 10.0, 15.0, and 25.0 μM) with a constant amount of MTs (5 μM tubulin). Dissociation constant values (K_d, μM) generated from the data in each of the curves are shown. (C) The subcellular localization pattern of the full-length MFP (GFP-MFP), truncated versions of MFP (C1, C2, N1, N2), and MFP lacking the PTS1 tripeptide (ΔSRM) fused to the carboxyl-terminus of GFP in onion epidermal cells. Faint MTs were occasionally evident in cells expressing the GFP-C1 fusion protein (arrow). Each image is a selected region from an individual cell. Bar, 10 μm.