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N V Raikhel

Publications and source records attributed to N V Raikhel.

At least 19 recordsLinked to original sources
PubMed2001

VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis.

Most plant cells are characterized by the presence of a large central vacuole that in differentiated cells accounts for more than 90% of the total volume. We have undertaken a genetic screen to look for mutants that are affected in the formation of vacuoles in plants. In this study, we report that inactivation of the Arabidopsis gene VACUOLELESS1 (VCL1) blocks vacuole formation and alters the pattern of cell division orientation and cell elongation in the embryo. Consistent with a role in vacuole biogenesis, we show that VCL1 encodes the Arabidopsis ortholog of yeast Vps16p. In contrast to yeast mutants that lack a vacuolar compartment but are viable and morphologically normal, loss of the plant vacuole leads to aberrant morphogenesis and embryonic lethality.

Alleles↗
PubMed2001

Interactions between syntaxins identify at least five SNARE complexes within the Golgi/prevacuolar system of the Arabidopsis cell.

The syntaxin family of soluble N-ethyl maleimide sensitive factor adaptor protein receptors (SNAREs) is known to play an important role in the fusion of transport vesicles with specific organelles. Twenty-four syntaxins are encoded in the genome of the model plant Arabidopsis thaliana. These 24 genes are found in 10 gene families and have been reclassified as syntaxins of plants (SYPs). Some of these gene families have been previously characterized, with the SYP2-type syntaxins being found in the prevacuolar compartment (PVC) and the SYP4-type syntaxins on the trans-Golgi network (TGN). Here we report on two previously uncharacterized syntaxin groups. The SYP5 group is encoded by a two-member gene family, whereas SYP61 is a single gene. Both types of syntaxins are localized to multiple compartments of the endomembrane system, including the TGN and the PVC. These two groups of syntaxins form SNARE complexes with each other, and with other Arabidopsis SNAREs. On the TGN, SYP61 forms complexes with the SNARE VTI12 and either SYP41 or SYP42. SYP51 and SYP61 interact with each other and with VTI12, most likely also on the TGN. On the PVC, a SYP5-type syntaxin interacts specifically with a SYP2-type syntaxin, as well as the SNARE VTI11, forming a SNARE complex likely involved in TGN-to-PVC trafficking.

Amino Acid Sequence↗
PubMed2001

Disruption of individual members of Arabidopsis syntaxin gene families indicates each has essential functions.

Syntaxins are a large group of proteins found in all eukaryotes involved in the fusion of transport vesicles to target membranes. Twenty-four syntaxins grouped into 10 gene families are found in the model plant Arabidopsis thaliana, each group containing one to five paralogous members. The Arabidopsis SYP2 and SYP4 gene families contain three members each that share 60 to 80% protein sequence identity. Gene disruptions of the yeast (Saccharomyces cerevisiae) orthologs of the SYP2 and SYP4 gene families (Pep12p and Tlg2p, respectively) indicate that these syntaxins are not essential for growth in yeast. However, we have isolated and characterized gene disruptions in two genes from each family, finding that disruption of individual syntaxins from these families is lethal in the male gametophyte of Arabidopsis. Complementation of the syp21-1 gene disruption with its cognate transgene indicated that the lethality is linked to the loss of the single syntaxin gene. Thus, it is clear that each syntaxin in the SYP2 and SYP4 families serves an essential nonredundant function.

Arabidopsis↗
PubMed2001

Characterization of a family of Arabidopsis genes related to xyloglucan fucosyltransferase1.

To understand primary cell wall assembly in Arabidopsis, we have focused on identifying and characterizing enzymes involved in xyloglucan biosynthesis. Nine genes (AtFUT2-10) were identified that share between 47% and 62% amino acid similarity with the xyloglucan-specific fucosyltransferase AtFUT1. Reverse transcriptase-PCR analysis indicates that all these genes are expressed. Bioinformatic analysis predicts that these family members are fucosyltransferases, and we first hypothesized that some may also be involved in xyloglucan biosynthesis. AtFUT3, AtFUT4, and AtFUT5 were expressed in tobacco (Nicotiana tabacum L. cv BY2) suspension culture cells, and the resulting proteins did not transfer fucose (Fuc) from GDP-Fuc to tamarind xyloglucan. AtFUT3, AtFUT4, and AtFUT5 were overexpressed in Arabidopsis plants. Leaves of plants overexpressing AtFUT4 or AtFUT5 contained more Fuc than wild-type plants. Stems of plants overexpressing AtFUT4 or AtFUT5 contained more xylose, less arabinose, and less galactose than wild-type plants. We suggest that the AtFUT family is likely to include fucosyltransferases important for the synthesis of wall carbohydrates. A targeted analysis of isolated cell wall matrix components from plants altered in expression of these proteins will help determine their specificity and biological function.

Amino Acid Sequence↗
PubMed2000

The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana.

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.

Arabidopsis↗
PubMed2000

Biochemical characterization and molecular cloning of an alpha-1,2-fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea.

Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-position of the galactosyl residue closest to the reducing end of the repeating subunit. This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at approximately 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight ( approximately 250 kDa), indicating that the active form is an oligomer. The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP. The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity.

Amino Acid Sequence↗
PubMed2000

Unique features of the plant vacuolar sorting machinery.

Multiple types of vacuoles can exist within the same plant cell, and different vesicle-trafficking pathways transport proteins to each of them. Recent work has identified proteins unique to each vacuole type, and the transport pathways have begun to be elucidated. Plant trafficking proteins are usually encoded by small gene families, the different members of which have distinct functions in the endomembrane system.

Arabidopsis↗
PubMed2000

AtVPS45 complex formation at the trans-Golgi network.

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.

Animals↗
PubMed2000

The Arabidopsis genome. An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors.

Many factors have been characterized as essential for vesicle trafficking, including a number of proteins commonly referred to as soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) components. The Arabidopsis genome contains a remarkable number of SNAREs. In general, the vesicle fusion machinery appears highly conserved. However, whereas some classes of yeast and mammalian genes appear to be lacking in Arabidopsis, this small plant genome has gene families not found in other eukaryotes. Very little is known about the precise function of plant SNAREs. By contrast, the intracellular localization of and interactions between a large number of plant SNAREs have been determined, and these data are discussed in light of the phylogenetic analysis.

Arabidopsis↗
PubMed1999

Plant importin alpha binds nuclear localization sequences with high affinity and can mediate nuclear import independent of importin beta.

Nuclear import of conventional nuclear localization sequence (NLS)-containing proteins initially involves recognition by the importin (IMP) alpha/beta heterodimer, where IMPalpha binds the NLS and IMPbeta targets the IMPalpha/NLS-containing protein complex to the nuclear pore. Here we examine IMPalpha from the plant Arabidopsis thaliana (At-IMPalpha), which exhibits nuclear envelope localization typical of IMPbeta rather than IMPalpha in other eukaryotic cell systems. We show that At-IMPalpha recognizes conventional NLSs of two different types with high affinity (K(d) of 5-10 nM), in contrast to mouse IMPalpha (m-IMPalpha), which exhibits much lower affinity (K(d) of 50-70 nM) and only achieves high affinity in the presence of m-IMPbeta. Unlike m-IMPalpha, At-IMPalpha is thus a high affinity NLS receptor in the absence of IMPbeta. Interestingly, At-IMPalpha was also able to bind with high affinity to NLSs recognized specifically by m-IMPbeta and not m-IMPalpha, including that of the maize transcription factor Opaque-2. Reconstitution of nuclear import in vitro indicated that in the absence of exogenous IMPbeta subunit but dependent on RanGDP and NTF2, At-IMPalpha was able to mediate nuclear accumulation to levels comparable with those mediated by m-IMPalpha/beta. Neither m-IMPalpha nor -beta was able to mediate nuclear import in the absence of the other subunit. At-IMPalpha's novel NLS recognition and nuclear transport properties imply that plants may possess an IMPalpha-mediated nuclear import pathway independent of IMPbeta in addition to that mediated by IMPalpha/beta.

Amino Acid Sequence↗
PubMed1999

Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis.

Cell walls are crucial for development, signal transduction, and disease resistance in plants. Cell walls are made of cellulose, hemicelluloses, and pectins. Xyloglucan (XG), the principal load-bearing hemicellulose of dicotyledonous plants, has a terminal fucosyl residue. A 60-kilodalton fucosyltransferase (FTase) that adds this residue was purified from pea epicotyls. Peptide sequence information from the pea FTase allowed the cloning of a homologous gene, AtFT1, from Arabidopsis. Antibodies raised against recombinant AtFTase immunoprecipitate FTase enzyme activity from solubilized Arabidopsis membrane proteins, and AtFT1 expressed in mammalian COS cells results in the presence of XG FTase activity in these cells.

Amino Acid Sequence↗
PubMed1999

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP.

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.

Adenosine Triphosphate↗