The components of the cellular machinery that accomplish the various complex and dynamic membrane fusion events that occur at the division plane during plant cytokinesis, including assembly of the cell plate, are not fully understood. The most well-characterized component, KNOLLE, a cell plate-specific soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptor (SNARE), is a membrane fusion machine component required for plant cytokinesis. Here, we show the plant ortholog of Cdc48p/p97, AtCDC48, colocalizes at the division plane in dividing Arabidopsis cells with KNOLLE and another SNARE, the plant ortholog of syntaxin 5, SYP31. In contrast to KNOLLE, SYP31 resides in defined punctate membrane structures during interphase and is targeted during cytokinesis to the division plane. In vitro-binding studies demonstrate that AtCDC48 specifically interacts in an ATP-dependent manner with SYP31 but not with KNOLLE. In contrast, we show that KNOLLE assembles in vitro into a large approximately 20S complex in an Sec18p/NSF-dependent manner. These results suggest that there are at least two distinct membrane fusion pathways involving Cdc48p/p97 and Sec18p/NSF that operate at the division plane to mediate plant cytokinesis. Models for the role of AtCDC48 and SYP31 at the division plane will be discussed.Plant cell division is completed by the highly dynamic process of de novo cell plate construction leading to the separation of two daughter cells. Formation of this unique cytokinetic organelle involves at least three key membrane fusion steps: (a) fusion of secretory vesicles across the division plane to form a membranous tubular-vesicular network, (b) consolidation of the tubular-vesicular network, and (c) fusion of the cell plate leading-edge with the original parental plasma membrane to complete division (Samuels et al., 1995). These distinct stages of cell plate biogenesis likely involve both heterotypic and homotypic membrane fusion events. In addition to the cell plate, the division plane contains an extensive endoplasmic reticulum (ER) network that has been suggested to function in the formation of the cell plate (Hepler, 1982). Assembly of cell plateassociated ER is likely to involve homotypic fusion of ER membrane within the division plane.Two homologous classes of ATPases associated with various cellular activities (AAA) proteins (Frö hlich, 2001), Sec18p N-ethylmaleimide-sensitive fusion protein (NSF) and Cdc48p/p97 (p97 is also known as VCP), regulate a variety of secretory membrane fusion processes (Acharya et al., 1995;Latterich et al., 1995;Rabouille et al., 1995). Monomers of Sec18p/NSF and Cdc48p/p97 consist of two Mg 2ϩ -dependent ATPase domains and an N-terminal substrate/adapter domain that assemble into biologically active ring-shaped oligohexamers (Peters et al., 1990; Hanson et al., 1997). Of these two AAA complexes, the biochemical function of Sec18p/NSF, which along with its cofactor, soluble NSF-attachment protein (␣-SNAP) regulates hetero-and homotypic membrane fusion, has...

p97/CDC48 is a highly abundant hexameric AAAATPase that functions as a molecular chaperone in numerous diverse cellular activities. We have identified an Arabidopsis UBX domain-containing protein, PUX1, which functions to regulate the oligomeric structure of the Arabidopsis homolog of p97/CDC48, AtCDC48, as well as mammalian p97. PUX1 is a soluble protein that co-fractionates with non-hexameric AtCDC48 and physically interacts with AtCDC48 in vivo. Binding of PUX1 to AtCDC48 is mediated through the UBX-containing C-terminal domain. However, disassembly of the chaperone is dependent upon the N-terminal domain of PUX1. These findings provide evidence that the assembly and disassembly of the hexameric p97/CDC48 complex is a dynamic process. This new unexpected level of regulation for p97/CDC48 was demonstrated to be critical in vivo as pux1 loss-of-function mutants display accelerated growth relative to wild-type plants. These results suggest a role for AtCDC48 and PUX1 in regulating plant growth.

CDC48/p97 is a conserved homohexameric AAA-ATPase chaperone required for a variety of cellular processes but whose role in the development of a multicellular model system has not been examined. Here, we have used reverse genetics, visualization of a functional Arabidopsis (Arabidopsis thaliana) CDC48 fluorescent fusion protein, and morphological analysis to examine the subcellular distribution and requirements for AtCDC48A in planta. Homozygous Atcdc48A T-DNA insertion mutants arrest during seedling development, exhibiting decreased cell expansion and displaying pleiotropic defects in pollen and embryo development. Atcdc48A insertion alleles show significantly reduced male transmission efficiency due to defects in pollen tube growth. Yellow fluorescent protein-AtCDC48A, a fusion protein that functionally complements the insertion mutant defects, localizes in the nucleus and cytoplasm and is recruited to the division mid-zone during cytokinesis. The pattern of nuclear localization differs according to the stage of the cell cycle and differentiation state. Inducible expression of an Atcdc48A Walker A ATPase mutant in planta results in cytokinesis abnormalities, aberrant cell divisions, and root trichoblast differentiation defects apparent in excessive root hair emergence. At the biochemical level, our data suggest that the endogenous steady-state protein level of AtCDC48A is dependent upon the presence of ATPase-active AtCDC48A. These results demonstrate that CDC48A/p97 is critical for cytokinesis, cell expansion, and differentiation in plants.Members of the AAA (ATPase associated with different cellular activities)-ATPase protein family are characterized by either one (type I) or two (type II) 220 to 250 amino acid ATPase domains containing both conserved Walker A and B motifs per protomer (Beyer, 1997;Neuwald et al., 1999). The ATPase domains elicit protein conformational changes upon the nucleotide binding, hydrolysis, and product release that is believed to be required for the function of the mechanochemical enzyme (Rouiller et al

The Xenopus Cripto-1 protein is confined to the cells of the animal hemisphere during early embryogenesis where it regulates the formation of anterior structures. Cripto-1 protein accumulates only in animal cells because cripto-1 mRNA in cells of the vegetal hemisphere is translationally repressed. Here, we show that the RNA binding protein, Bicaudal-C (Bic-C), functioned directly in this vegetal cell-specific repression. While Bic-C protein is normally confined to vegetal cells, ectopic expression of Bic-C in animal cells repressed a cripto-1 mRNA reporter and associated with endogenous cripto-1 mRNA. Repression by Bic-C required its N-terminal domain, comprised of multiple KH motifs, for specific binding to relevant control elements within the cripto-1 mRNA and a functionally separable C-terminal translation repression domain. Bic-C-mediated repression required the 5 ′ CAP and translation initiation factors, but not a poly(A) tail or the conserved SAM domain within Bic-C. Bic-C-directed immunoprecipitation followed by deep sequencing of associated mRNAs identified multiple Bic-C-regulated mRNA targets, including cripto-1 mRNA, providing new insights and tools for understanding the role of Bic-C in vertebrate development.

No abstract