Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

SUN–KASH nuclear envelope bridges formed by WIP and SUN proteins are present in the plant branch of the tree of life but have functionally diverged from their opisthokont counterparts and are involved in nuclear morphology and RanGAP–nuclear envelope association.

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A) þ RNA. nua mutants phenocopy mutants of EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and additional developmental processes.

Ran is a small signaling GTPase that is involved in nucleocytoplasmic transport. Two additional functions of animal Ran in the formation of spindle asters and the reassembly of the nuclear envelope in mitotic cells have been recently reported. In contrast to Ras or Rho, Ran is not associated with membranes. Instead, the spatial sequestering of its accessory proteins, the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1, appears to define the local concentration of RanGTP vs. RanGDP involved in signaling. Mammalian RanGAP is bound to the nuclear pore by a mechanism involving the attachment of small ubiquitin-related modifier protein (SUMO) to its C terminus and the subsequent binding of the SUMOylated domain to the nucleoporin Nup358. Here we show that plant RanGAP utilizes a different mechanism for nuclear envelope association, involving a novel targeting domain that appears to be unique to plants. The N-terminal WPP domain is highly conserved among plant RanGAPs and the small, plant-specific nuclear envelope-associated protein MAF1, but not present in yeast or animal RanGAP. Confocal laser scanning microscopy of green fluorescent protein (GFP) fusion proteins showed that it is necessary for RanGAP targeting and sufficient to target the heterologous protein GFP to the plant nuclear rim. The highly conserved tryptophan and proline residues of the WPP motif are necessary for its function. The 110-aa WPP domain is the first nuclear-envelope targeting domain identified in plants. Its fundamental difference to its mammalian counterpart implies that different mechanisms have evolved in plants and animals to anchor RanGAP at the nuclear surface.

In higher plants, the plane of cell division is faithfully predicted by the preprophase band (PPB). The PPB, a cortical ring of microtubules and F-actin, disassembles upon nuclear-envelope breakdown. During cytokinesis, the expanding cell plate fuses with the plasma membrane at the cortical division site, the site of the former PPB. The nature of the ''molecular memory'' that is left behind by the PPB and is proposed to guide the cell plate to the cortical division site is unknown. RanGAP is the GTPase activating protein of the small GTPase Ran, which provides spatial information for nucleocytoplasmic transport and various mitotic processes in animals. Here, we show that, in dividing root cells, Arabidopsis RanGAP1 concentrates at the PPB and remains associated with the cortical division site during mitosis and cytokinesis, requiring its N-terminal targeting domain. In a fass/ton2 mutant, which affects PPB formation, RanGAP1 recruitment to the PPB site is lost, while its PPB retention is microtubule-independent. RanGAP1 persistence at the cortical division site, but not its initial accumulation at the PPB requires the 2 cytokinesis-regulating kinesins POK1 and POK2. Depletion of RanGAP by inducible RNAi leads to oblique cell walls and cell-wall stubs in root cell files, consistent with cytokinesis defects. We propose that Arabidopsis RanGAP, a continuous positive protein marker of the plant division plane, has a role in spatial signaling during plant cell division.cytokinesis ͉ Ran cycle ͉ preprophase band ͉ nuclear pore

The Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, and nuclear envelope (NE) formation [1-4]. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1 [5-8]. Mammalian RanGAP1 targeting to the NE and kinetochores requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2 [9-14]. In contrast, Arabidopsis RanGAP1 is associated with the NE and cell plate, mediated by an N-terminal, plant-specific WPP domain [15-18]. In the absence of RanBP2 in plants, the mechanism for spatially sequestering plant RanGAP is unknown. Here, Arabidopsis WPP-domain interacting proteins (WIPs) that interact with RanGAP1 in vivo and colocalize with RanGAP1 at the NE and cell plate were identified. Immunogold labeling indicates that WIP1 is associated with the outer NE. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root-tip cells, whereas NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIPs are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms.

Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems, motors, levers, rotating arms and possibly springs. A growing number of human diseases are found to be caused by mutations in long coiled-coil proteins. This review summarizes our current understanding of the multifaceted group of long coiled-coil proteins in the cytoskeleton, nucleus, Golgi and cell division apparatus. The biophysical features of coiled-coil domains provide first clues toward their contribution to the diverse protein functions and promise potential future applications in the area of nanotechnology. Combining the power of fully sequenced genomes and structure prediction algorithms, it is now possible to comprehensively summarize and compare the complete inventory of coiled-coil proteins of different organisms.

SummaryThe RBCS3A gene of tomato belongs to a small gene family consisting of ®ve members. Although the RBCS1, RBCS2 and RBCS3A promoters contain closely related cis regulatory sequences, the expression patterns of the genes are different. Whereas the RBCS1 and RBCS2 genes are expressed in both leaves and young fruit, the RBCS3A promoter is highly active in leaves, but not in young fruit. This lack of transcription could be due to a mutation in the RBCS3A promoter creating the so-called F-box, a protein binding site located between the activating cis elements, the I-box and G-box. In order to identify proteins that bind to the RBCS3A I-box/F-box region, the yeast one-hybrid system was used. One clone, LeMYBI was isolated which contains strong similarity to plant myb transcription factors. The encoded LeMYBI protein is at least 188 amino acids in length and contains two myb-like domains located at the amino terminus and close to the carboxy terminus, separated by a negatively charged domain. The protein contains a SHAQKYF amino acid signature motif in the second myb-like repeat, which is highly conserved in a number of recently identi®ed plant myb-related genes, thus de®ning a new class of plant DNA-binding proteins. LeMYBI binds speci®cally to the I-box sequence of the RBCS1, RBCS2 and RBCS3A promoters, therefore representing the ®rst cloned I-box binding factor. LeMYBI acts as a transcriptional activator in yeast and plants, and binds to the I-box with a DNA-binding domain located in the carboxyterminal domain.