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N V Raikhel

Publications and source records attributed to N V Raikhel.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1999

The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment.

Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.

Amino Acid Sequence↗
PubMed1999

The t-SNARE AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells.

Protein cargo is trafficked between the organelles of the endomembrane system inside transport vesicles, a process mediated by integral membrane proteins called SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) that reside on the surface of the vesicle (v-SNAREs) and target membrane (t-SNAREs). In examining transport of cargo between the trans-Golgi network and the vacuole in Arabidopsis, we have previously characterized AtPEP12p as a t-SNARE residing on the prevacuolar compartment and AtVTI1a as a v-SNARE that interacts with AtPEP12p. Recently, we have begun to characterize AtVAM3p, another Arabidopsis t-SNARE that shows high sequence homology to AtPEP12p. We have found that AtVTI1a also interacts with AtVAM3p, suggesting a role for this t-SNARE in post-Golgi trafficking. AtVAM3p has been suggested to localize to the vacuolar membrane in Arabidopsis cells; however, using specific antisera and expression of epitope-tagged versions of each t-SNARE, we have discovered that AtVAM3p is found on the same prevacuolar structure as AtPEP12p in Arabidopsis root cells.

Arabidopsis↗