A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

Abstract

The microtubules (MTs) of higher plant cells a= organized into arrays with essential functions in plant cell growth and differentiation; however, molecular mechanisms underlying the organization and regulation of these arrays remain largely unknown. We have approached this problem using tubulin affinity chromatography to isolate carrot proteins that interact with MTs. From these proteins, a 50-kD polypeptide was selectively purified by exploiting its Ca2+-dependent binding to calmodulin (CaM). This polypeptide was identified as a homolog of elongation factor-lu (EF-1a)-a highly conserved and ubiqultous protein translation factor. The carrot EF-la homolog bundles MTs in vitro, and moreover, this bundling is modulated by the addition of Ca2+ and CaM together (Ca2+/CaM). A direct binding between the EF-la homolog and MTs was demonstrated, providing nove1 evidence for such an interaction. Based on these findings, and others discussed herein, we propose that an EF-la homolog mediates the lateral association of MTs in plant celk by a Ca2+/CaM-sensitive mechanism. INTRODUCTIONMicrotubules (MTs) participate in a number of essential processes in eukaryotes (MacRae, 1992b). In cells of higher plants, MTs are generally arranged into four structurally and functionally distinct, cell cycle-dependent MT arrays (Goddard et al., 1994;Lambert and Lloyd, 1994). During interphase, the MT array in a cell's cortex is involved in the extracellular deposition of cellulose microfibrils (Giddings and Staehelin, 1991). During G2 of the cell cycle, this array is succeeded by a narrower preprophase band involved in establishing the site of the incipient division plane (Palevitz, 1991; Wick, 1991). The preprophase band resolves into the mitotic spindle apparatus as M phase progresses Palevitz, 1993). The fourth MT array, the phragmoplast, appears as cytokinesis begins and is involved in depositing the new cell plate at the position previously marked by the preprophase band (Lloyd, The bases by which plant MTs are organized into higher order arrays and by which those arrays are regulated remain largely unknown (Lambert, 1993). MTs are hollow cylinders with 25-nm diameters, and tubulin is the principal protein subunit of MTs. MTs assembled from pure tubulin have a limited range of behavior-they assemble or disassemble. This limited range of behavior can only partly account for the diverse structures and functions of MT arrays.Nearly all species have multiple tubulin genes encoding various tubulin isotypes (for plant genes, see Goddard et al., 1994), and it has been proposed that the isotypes may provide a 1991).To whom correspondence should be addressed. means for serving different MT functions (Fulton and Simpson, 1976). However, nearly all existing evidence indicates that tubulin isotypes are functionally interchangeable (LudueAa, 1993). In general, tubulins are COnseNed even between kingdoms (Fosket and Morejohn, 1992; Burns and Surridge, 1994). In fact, animal brain tubulin that is microinjected into living plant cells incorporates into t...

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

“…Previously, we demonstrated that bovine brain tubulin affinity columns were as effective in purifying rice tubulin-binding proteins as were columns generated with rice tubulin as the ligand (5). In addition, tubulin and MT affinity chromatography of plant extracts resulted in the purification of proteins that had SDS-PAGE profiles that were qualitatively similar (5,13). The ease of purifying large amounts of bovine brain tubulin and the enhanced stability of tubulin columns over MT columns prompted us to use bovine tubulin affinity columns in this study, as in our previous study (5).…”

“…Two studies demonstrated that the SDS-PAGE profiles of plant proteins that eluted from tubulin and MT affinity chromatography columns were qualitatively similar (5,13), and essentially all proteins that bound to the tubulin affinity columns subsequently co-sedimented with MTs in pelleting assays (14). This indicates that tubulin affinity chromatography can be used to successfully purify MT-binding proteins.…”

The Nuclear Migration Protein NUDF/LIS1 Forms a Complex with NUDC and BNFA at Spindle Pole Bodies

“…The upper band was identified as NUDF (AN6197.1; two tryptic peptides of the protein with cross correlation [Xcorr] values of 3.5 and 3.9) and the band below as the elongation factor 1A ␣-subunit (AN4218.1; three peptides with Xcorr values of 2.3, 3.0, and 3.4). The elongation factor also was identified in other control purifications, and since it was shown to bind to calmodulin (11), its copurification in this experiment probably was not due to a specific binding to NUDF. The lower band contained the gene product of ORF AN3213.1 (only one peptide, with an Xcorr value of 2.8), which we called BNFA.…”

The Characterization of Plasma Membrane-Bound Tubulin of Cauliflower Using Triton X-114 Fractionation

“…There are several examples of MAPs associated with cortical MTs, but so far it is not known whether they constitute the actual link between MTs and the PM. Examples of plant MAPs include a 76-kD MAP of carrot (Daucus carota) suspension cells (Cyr and Palevitz, 1989), an 83-kD maize protein immunologically related to T (Vantard et al, 1991), the elongating factor 1-a in carrot suspension cells (Durso and Cyr, 1994), and a 65-kD MAP of tobacco (Nicofiana tabacum) BY-2 cells (Chang-Jie and Sonobe, 1993).…”