Biochemical Purification Approaches

Co-Assembly and Co-Purification of MT-Binding Proteins Using Endogenous and Neuronal MTs. The ability to assemble tubulin dimers into MTs in vitro has been the basis for protocols that involve co-assembly or co-purification of MT-binding proteins with MTs. Protocols of this type have been typically modeled after those used for animal cells [16]. Preformed MTs, or MTs assembled directly from endogenous tubulin in plant cell extracts, are used as a matrix onto which MT-binding proteins are bound in vitro. MTs are then pelleted by centrifugation, and the associated proteins are released by high-ionic-strength treatment. Owing to the low concentration of tubulin in most types of plant cells, mammalian neuronal tubu-lin is often used as the source of MTs in many plant MAP purification schemes. Brain tissue is rich in MTs, and standard protocols are available for tubulin purification from this tissue [17]. However, several reports have described the purification of MT-binding proteins using endogenous plant MTs assembled directly in cell extracts in the presence of the MT-stabilizing agent taxol [18-21]. A strategy to further enrich MT-binding proteins using this approach involves adding cycles of MT depolymeriza-tion and repolymerization to the pellet fraction [18]. Korolev et al. [22] demonstrated that the line of cell suspension used was essential for successful MT-binding protein purification with endogenous plant MTs. They concluded that small, dense cells of an Arabidopsis Landsberg cell line proved more effective for this procedure than the larger, vacuolated cells common to cell lines from other genotypes. Readers are directed elsewhere for a detailed description of methods used to purify MT-binding proteins via co-assembly of endogenous plant MTs [18, 20, 22] or by co-purification with exogenous neuronal MTs [23, 24].

MT- and Tubulin-Affinity Chromatography (MAC and TAC). MAC and

TAC have proven effective as approaches for purifying authentic MT-binding proteins from a wide range of species. Protocols for column preparation and chromatography were based on those established for the isolation of Drosophila MT-binding proteins [25, 26]. Taxol-stabilized MTs or native tubulin dimers are covalently coupled to a chromatography matrix that often consists of activated agarose beads. A BSA column serves as a useful control column that can identify nonspecific interactions, since the overall pI of BSA is similar to that of tubulin. Clarified cell extracts are passed slowly over the column matrices, followed by column washing and then elution of bound proteins with solutions containing high concentrations of salt.

Interestingly, profiles of plant proteins that eluted from MAC and TAC columns were qualitatively similar when analyzed by SDS-PAGE, regardless of whether plant or neuronal tubulin was used as the source of tubulin [27, 28] (Figure 19.1A). Approximately 30-50 proteins are visible in these fractions when resolved by 1D SDS-PAGE slab gels and silver staining (Figure 19.1A). However, the complexity of proteins is more readily visible using 2-DGE and CBB [4, 28] or silver staining procedures (Chuong and Muench, unpublished observations). Indeed, well in excess of 100 proteins are clearly visible on 2D gels (Figure 19.1B), and numerous, more faintly staining protein spots are also present. We found that resolving these affinity chromatogra-phy purified proteins using 2-DGE required the use of NEPHGE techniques [29]. MT-binding proteins generally have a basic pI [28, 30], causing them to precipitate using the standard first-dimensional gel systems. However, this problem was overcome using NEPHGE. Here, the protein samples are loaded in tube gels that are immersed in acidic running buffer, thereby limiting the precipitation of this basic protein fraction [4, 28, 31].

The similarity between the SDS-PAGE profiles of proteins eluted from MT and TAC columns demonstrates that many proteins bind nearly as effectively to tubu-lin dimers as they do to MTs. Indeed, MT pelleting assays using tubulin-binding protein fractions indicated that a majority of the proteins in this fraction also bind

FIGURE 19.1. Protein profiles of MT- and tubulin-affinity chromatography-purified protein fractions, along with the class representations of MT-binding proteins from proteomic studies. (A) Silver-stained SDS-PAGE profiles of rice endosperm proteins that eluted from MT- or tubulin-affinity chromatography columns prepared using plant or bovine tubulin. Fractions were loaded on the columns in the presence of 50 mM KCl, washed, and eluted with 500 mM KCl. A BSA affinity column served as the control. (From reference 28 with permission from the publishers.) (B) CBB-stained 2D NEPHGE gel of an Arabidopsis cell suspension protein fraction eluted from a bovine tubulin-affinity column. The approximate pH value at specific locations in the first dimension gel is indicated at the top of the gel. Molecular mass standards are listed on the left of the gel. Candidate spots selected for LC-MS/MS analysis are numbered. (From reference 4 with permission from the publishers.) (C) Classes of proteins identified in a large-scale tubulin-affinity chromatography study. (From reference 4 with permission from the publishers.) (D) Classes of proteins identified in a large-scale MT co-assembly study [22]. Several tubulin isotypes were identified in this study, but were not included in this figure.

FIGURE 19.1. Protein profiles of MT- and tubulin-affinity chromatography-purified protein fractions, along with the class representations of MT-binding proteins from proteomic studies. (A) Silver-stained SDS-PAGE profiles of rice endosperm proteins that eluted from MT- or tubulin-affinity chromatography columns prepared using plant or bovine tubulin. Fractions were loaded on the columns in the presence of 50 mM KCl, washed, and eluted with 500 mM KCl. A BSA affinity column served as the control. (From reference 28 with permission from the publishers.) (B) CBB-stained 2D NEPHGE gel of an Arabidopsis cell suspension protein fraction eluted from a bovine tubulin-affinity column. The approximate pH value at specific locations in the first dimension gel is indicated at the top of the gel. Molecular mass standards are listed on the left of the gel. Candidate spots selected for LC-MS/MS analysis are numbered. (From reference 4 with permission from the publishers.) (C) Classes of proteins identified in a large-scale tubulin-affinity chromatography study. (From reference 4 with permission from the publishers.) (D) Classes of proteins identified in a large-scale MT co-assembly study [22]. Several tubulin isotypes were identified in this study, but were not included in this figure.

to MTs in vitro [4, 32]. TAC has been used to identify several authentic plant and animal MT-binding proteins [27, 28, 32, 33]. However, there are examples of structural MAPs that do not bind to tubulin dimers [34] and thus would not be purified using TAC. Close inspection of SDS-PAGE protein profiles showed that subtle differences are recognizable between the fractions purified by MAC versus TAC techniques

(Figure 19.1A). However, the high yield of tubulin in brain tissue extracts and the stability of TAC columns have made neuronal TAC an appealing approach for our laboratory.

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