Enzyme screens with Strep-tagged recombinant proteins and expression studies with the respective green fluorescent protein (GFP) fusions have been employed to examine the functional activities and subcellular localization of members of the Arabidopsis glutathione transferase (GST) superfamily. Fifty-one of 54 GST family members were transcribed and 41 found to express as functional glutathione-dependent enzymes in Escherichia coli. Functional redundancy was observed and in particular three theta (T) class GSTs showed conserved activities as hydroperoxide-reducing glutathione peroxidases (GPOXs). When expressed in tobacco as GFP fusions, all three GSTTs localized to the peroxisome, where their GPOX activity could prevent membrane damage arising from fatty acid oxidation. Through alternative splicing, two of these GSTTs form fusions with Myb transcription factor-like domains. Examination of one of these variants showed discrete localization within the nucleus, possibly serving a role in reducing nucleic acid hydroperoxides or in signalling. Based on this unexpected differential sub-cellular localization, 15 other GST family members were expressed as GFP fusions in tobacco. Most accumulated in the cytosol, but GSTU12 localized to the nucleus, a family member resembling a bacterial tetrachlorohydroquinone dehalogenase selectively associated with the plasma membrane, and a lambda GSTL2 was partially directed to the peroxisome after removal of a putative chloroplast transit peptide. Based on the results obtained with the GSTTs, it was concluded that these proteins can exert identical protective functions in differing subcellular compartments.

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

MOR1 is a member of the MAP215 family of microtubule-associated proteins and is required to establish interphase cortical microtubule arrays in plant cells.1 Here we show that MOR1 binds microtubules in vivo, localising to both cortical microtubules and to areas of overlapping microtubules in the phragmoplast. We demonstrate an essential role for MOR1 in cytokinesis through genetic complementation of the cytokinesis defective gem1-1 mutation with MOR1. Phenotypic analysis of gem1-1 and a T-DNA insertion allele gem1-2 confirm that MOR1/GEM1 is essential for regular patterns of cytokinesis. Both mutations, gem1-1 and gem1-2, cause the truncation of MOR1/GEM1. Moreover, the C-terminal domain absent in both mutants binds microtubules in vitro. These data demonstrate that MOR1/GEM1 plays an essential role in the cytokinetic phragmoplast. TextIn general microtubules form four distinct microtubule assemblies sequentially through the plant cell cycle. The interphase cortical array is involved in cell expansion. The preprophase band of microtubules delineates the plane of cell division. The spindle separates daughter chromosomes, and the phragmoplast, which forms in late anaphase, guides golgi-derived vesicles to a site where they will fuse to form the new cell plate that separates the daughter cells. This pattern of alternating arrays is reflected in every cell division in the plant, with the exception of meiotic cell divisions and subsequent gametophytic and endosperm mitoses, all of which lack a preprophase band. As in animal cells microtubule associated proteins must govern the organisation of these microtubule arrays. Three classes of plant structural MAP have been identitied two of which are unique to plants, MAP-652 and MAP1903,4, and the third is a homologue of Xenopus MAP215, named MOR11,5.The MAP215 family also includes human Ch-TOGp6, Dictyostelium discoideum DdCP2247, Drosophila melanogaster Msps8, Caenorhabditis elegans Zyg-99,

Complex animals use a wide variety of adaptor proteins to produce specialized sites of interaction between actin and membranes. Plants do not have these protein families, yet actin-membrane interactions within plant cells are critical for the positioning of subcellular compartments, for coordinating intercellular communication, and for membrane deformation. Novel factors are therefore likely to provide interfaces at actin-membrane contacts in plants, but their identity has remained obscure. Here we identify the plant-specific Networked (NET) superfamily of actin-binding proteins, members of which localize to the actin cytoskeleton and specify different membrane compartments. The founding member of the NET superfamily, NET1A, is anchored at the plasma membrane and predominates at cell junctions, the plasmodesmata. NET1A binds directly to actin filaments via a novel actin-binding domain that defines a superfamily of thirteen Arabidopsis proteins divided into four distinct phylogenetic clades. Members of other clades identify interactions at the tonoplast, nuclear membrane, and pollen tube plasma membrane, emphasizing the role of this superfamily in mediating actin-membrane interactions.

Summary The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant‐specific NETWORKED protein, NET3C, and phylogenetically conserved vesicle‐associated membrane protein‐associated proteins (VAPs). Ten VAP homologues (VAP27‐1 to 27‐10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27‐labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27‐labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27‐GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER–cytoskeleton interaction and for plant development.