Purification and Characterization of Novel Microtubule-Associated Proteins from Arabidopsis Cell Suspension Cultures1[C][W]

Plant microtubules (MTs) play essential roles in cell division, anisotropic cell expansion, and overall organ morphology. Microtubule-associated proteins (MAPs) bind to MTs and regulate their dynamics, stability, and organization. Identifying the full set of MAPs in plants would greatly enhance our understanding of how diverse MT arrays are formed and function; however, few proteomics studies have characterized plant MAPs. Using liquid chromatography-tandem mass spectrometry, we identified hundreds of proteins from MAP-enriched preparations derived from cell suspension cultures of Arabidopsis (Arabidopsis thaliana). Previously reported MAPs, MT regulators, kinesins, dynamins, peroxisome-resident enzymes, and proteins implicated in replication, transcription, and translation were highly enriched. Dozens of proteins of unknown function were identified, among which 12 were tagged with green fluorescent protein (GFP) and examined for their ability to colocalize with MTs when transiently expressed in plant cells. Six proteins did indeed colocalize with cortical MTs in planta. We further characterized one of these MAPs, designated as BASIC PROLINE-RICH PROTEIN1 (BPP1), which belongs to a sevenmember family in Arabidopsis. BPP1-GFP decorated interphase and mitotic MT arrays in transgenic Arabidopsis plants. A highly basic, conserved region was responsible for the in vivo MT association. Overexpression of BPP1-GFP stabilized MTs, caused right-handed helical growth in rapidly elongating tissues, promoted the formation of transverse MT arrays, and resulted in the outgrowth of epidermal cells in light-grown hypocotyls. Our high-quality proteome database of Arabidopsis MAP-enriched preparations is a useful resource for identifying novel MT regulators and evaluating potential MT associations of proteins known to have other cellular functions.