Purification and Characterization of Novel Microtubule-Associated Proteins from Arabidopsis Cell Suspension Cultures    

Abstract

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.Microtubules (MTs) are major structural components of the plant cytoskeleton that are composed of a-tubulin/b-tubulin heterodimer subunits and are intricately involved in cell division, morphology, and intracellular organization and transport. The ability of the MT cytoskeleton to fulfill its versatile cellular functions relies on its intrinsically dynamic polymer properties. Individual MTs alternate between phases of growth and shrinkage by rapidly attaching and removing tubulin subunits at their ends. These cycles of polymerization and depolymerization, which continuously reorganize the MT cytoskeleton, are sometimes interrupted by pauses in which neither growth nor shrinkage occurs (Desai and Mitchison,

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus

“…6, H and I). Consistent with previous reports (Hamada et al, 2013), MTs in wild-type (GFP-MAP4) hypocotyl cells were distributed randomly. However, in IQD16 (GFP-MAP4) and GFP-IQD16-overexpressing lines, MTs decorated by the GFP reporter were aligned preferentially in oblique arrays with angles between 40°and 70° (Fig.…”

“…A phosphoproteome analysis of PM-associated proteins after elicitor treatment identified several IQDs in PMenriched fractions, including IQD9, IQD11, and IQD14 (Benschop et al, 2007). Interrogation of the nuclear proteome detected IQD28 (Bigeard et al, 2014;Palm et al, 2016) and IQD32 (Bigeard et al, 2014), which independently confirms the targeting of at least two IQD proteins to multiple subcellular sites (Hamada et al, 2013).…”

“…Analysis of MAP-enriched fractions prepared from Arabidopsis cell cultures identified a large set of known and novel MAPs. Interestingly, four IQD proteins of phylogenetic group IV (IQD28, IQD29, IQD31, and IQD32) were highly enriched in these fractions, and MT colocalization was confirmed for GFP fusions of IQD28 and IQD32 (Hamada et al, 2013). A phosphoproteome analysis of PM-associated proteins after elicitor treatment identified several IQDs in PMenriched fractions, including IQD9, IQD11, and IQD14 (Benschop et al, 2007).…”

Roles of MAP65-1 and BPP1 in Gravity Resistance of Arabidopsis hypocotyls

“…The construct was introduced into Arabidopsis by an Agrobacterium (Rhizobium radiobacter, EHA105 strain)-mediated method. As to BPP1-GFP, the details were described previously (Hamada et al, 2013).…”

“…Microtubule-associated proteins (MAPs) bind to microtubules and regulate their dynamics, stability, and organization (Gardiner, 2013;Hamada, 2014). It is reported that 65 kDa microtubuleassociated proteins (MAP65s) and basic proline-rich protein1 (BPP1) are required for the maintenance of transverse microtubule orientation (Sawano et al, 2000;Hamada et al, 2013). We produced green fluorescent protein (GFP) lines in which GFP was fused with these MAPs in Arabidopsis.…”