The sponge-derived antimitotic tripeptide hemiasterlin was previously shown to inhibit tubulin polymerization. We have now demonstrated that hemiasterlin resembles most other antimitotic peptides in noncompetitively inhibiting the binding of vinblastine to tubulin (apparent K(i) value, 7.0 microM), competitively inhibiting the binding of dolastatin 10 to tubulin (apparent K(i) value, 2.0 microM), stabilizing the colchicine binding activity of tubulin, inhibiting nucleotide exchange on beta-tubulin, and inducing the formation of tubulin oligomers that are stable to gel filtration in the absence of free drug, even at low drug concentrations. The tubulin oligomerization reaction induced by hemiasterlin was compared to the reactions induced by dolastatin 10 and cryptophycin 1. Like dolastatin 10, hemiasterlin induced formation of a tubulin aggregate that had the morphological appearance primarily of ring-like structures with a diameter of about 40 nm, while the morphology of the cryptophycin 1 aggregate consisted primarily of smaller rings (diameter about 30 nm). However, the hemiasterlin aggregate differed from the dolastatin 10 aggregate in that its formation was not associated with turbidity development, and the morphology of the hemiasterlin aggregate (as opposed to the dolastatin 10 aggregate) did not change greatly when microtubule-associated proteins were present (tight coils and pinwheels are observed with dolastatin 10 but not with hemiasterlin or cryptophycin 1). Opacification of tubulin-dolastatin 10 mixtures was inhibited by hemiasterlin at 22 degrees C and stimulated at 0 degrees C, while cryptophycin 1 was inhibitory at both reaction temperatures.

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...

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The microtubules (MTs) of higher plant cells are 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 Ca(2+)-dependent binding to calmodulin (CaM). This polypeptide was identified as a homolog of elongation factor-1 alpha (EF-1 alpha)--a highly conserved and ubiquitous protein translation factor. The carrot EF-1 alpha 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-1 alpha homolog and MTs was demonstrated, providing novel evidence for such an interaction. Based on these findings, and others discussed herein, we propose that an EF-1 alpha homolog mediates the lateral association of MTs in plant cells by a Ca2+/CaM-sensitive mechanism.

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The organization and function of microtubules in plant cells are important in key developmental events, including the regulation of directional cellulose deposition. Bridges connecting microtubules to each other and to membranes and other organelles have been documented by electron microscopy; however, the biochemical and molecular nature of these linkages is not known. We have partitioned proteins from a suspension culture of tobacco into cytosolic and membrane fractions, solubilized the membrane fraction with a zwitterionic detergent, and then used affinity chromatography and salt elution to isolate tubulin binding proteins. Dark-field microscopy of in vitro-assembled microtubules showed that the eluted proteins from both fractions induce microtubule bundling and, in the presence of purified tubulin, promote microtubule elongation. Gel electrophoresis of the eluted proteins revealed two distinct sets of polypeptides. Those in the membrane eluate included unique bands with apparent molecular masses of 98, 90, and 75 kD in addition to bands present in both eluates. The cytosolic eluate, in contrast, typically included relatively smaller proteins. The eluted proteins also bound to taxol-stabilized microtubules. Initial immunological characterization using monoclonal antibodies raised against the 90-kD polypeptide showed that it is colocalized in situ with cortical microtubules in tobacco protoplast ghosts.

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