Showing papers by "Natasha V. Raikhel published in 1998"
TL;DR: It is found that AtELP is located at the trans-Golgi of Arabidopsis root cells, and that its C terminus can preferentially interact in vitro with the mammalian TGN-specific AP-1 clathrin-adapter complex, suggesting a likely role in clathin-coated, vesicle-directed trafficking at the TGN.
Abstract: Targeting of protein cargo to the vacuole/lysosome is a multistep process that appears to have conserved features between mammalian, yeast, and plant cells. In each case, some soluble vacuolar/lysosomal proteins are believed to be bound by transmembrane cargo receptors in the trans-Golgi network (TGN) that redirect these proteins into clathrin-coated vesicles. These vesicles then appear to be transported to the prevacuole/endosome by a trafficking machinery that requires components identified in other vesicle-targeting steps such as N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), SNAP receptors (SNAREs), rab-type GTPases, and Sec1p homologs. Two likely members of this trafficking machinery have been characterized from Arabidopsis thaliana: AtPEP12p, a t-SNARE that resides on a what we now call a prevacuolar compartment, and AtELP, a protein that shares many common features with mammalian and yeast transmembrane cargo receptors. Here, we have further investigated the intracellular distribution of AtELP. We have found that AtELP is located at the trans-Golgi of Arabidopsis root cells, and that its C terminus can preferentially interact in vitro with the mammalian TGN-specific AP-1 clathrin–adapter complex, suggesting a likely role in clathrin-coated, vesicle-directed trafficking at the TGN. Further, consistent with a role in trafficking of vacuolar cargo, we have found that AtELP partially colocalizes with AtPEP12p on a prevacuolar compartment.
173 citations
TL;DR: It is confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates.
Abstract: A reversibly glycosylated polypeptide from pea ( Pisum sativum ) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of A rabidopsis t haliana, Reversibly GlycosylatedPolypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.
79 citations
TL;DR: Biochemical analysis showed that importin α associated with microtubules and microfilaments in vitro in an NLS-dependent manner, suggesting that the interaction of importinα with the cytoskeleton could be an essential element of protein transport from the cytoplasm to the nucleus in vivo.
Abstract: Importin α is the nuclear localization signal (NLS) receptor that is involved in the nuclear import of proteins containing basic NLSs. Using importin α as a tool, we were interested in determining whether the cytoskeleton could function in the transport of NLS-containing proteins from the cytoplasm to the nucleus. Double-labeling immunofluorescence studies showed that most of the cytoplasmic importin α coaligned with microtubules and microfilaments in tobacco protoplasts. Treatment of tobacco protoplasts with microtubule- or microfilament-depolymerizing agents disrupted the strands of importin α in the cytoplasm, whereas a microtubule-stabilizing agent had no effect. Biochemical analysis showed that importin α associated with microtubules and microfilaments in vitro in an NLS-dependent manner. The interaction of importin α with the cytoskeleton could be an essential element of protein transport from the cytoplasm to the nucleus in vivo.
73 citations
TL;DR: Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations.
Abstract: The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system. This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae). We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45). The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related. AtVPS45p is a peripheral membrane protein that associates with microsomal membranes. Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations. These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants.
58 citations
TL;DR: The nuclear pore complex is the largest supramolecular complex that assembles in the eukaryotic cell and must disassemble prior to mitosis and reassemble after the event, a potentially regulatory point for translocation.
Abstract: The nuclear pore complex is the largest supramolecular complex that assembles in the eukaryotic cell. This structure is highly dynamic and must disassemble prior to mitosis and reassemble after the event. The directed movement of macromolecules into and out of the nucleus occurs through the nuclear pore complex, a potentially regulatory point for translocation. Using biochemical and genetic approaches, several nuclear pore complex proteins from yeast and vertebrates have been well characterized. Although very little is known about plant nuclear pore proteins, research is providing new information that indicates that plant nuclear pore complexes may have some unique features.
43 citations
TL;DR: Peptide sequence information derived from one tGlcNAc protein with an apparent molecular mass of 40 to 43 kD made it possible to clone its gene and gp40 was localized to the nuclear rim, as shown by biochemical fractionation and immunofluorescence microscopy.
Abstract: Several glycoproteins that are present at the nuclear rim and at the nuclear pore complex of tobacco suspension-cultured cells are modified by O-linked oligosaccharides with terminal N-acetylglucosamine (GlcNAc). Here, we report on the purification of several of these glycoproteins, which are referred to as terminal GlcNAc (tGlcNAc) proteins. In vitro galactosylation of the tGlcNAc proteins generated glycoproteins with terminal galactosyl-beta-1, 4-GlcNAc and thus permitted their isolation by Erythrina crystagalli agglutinin affinity chromatography. Peptide sequence information derived from one tGlcNAc protein with an apparent molecular mass of 40 to 43 kD, designated gp40, made it possible to clone its gene. Interestingly, gp40 has 28 to 34% amino acid identity to aldose-1-epimerases from bacteria, and no gene encoding an aldose-1-epimerase has been isolated previously from higher organisms. Polyclonal antibodies were generated against recombinant gp40. Consistent with its purification as a putative nuclear pore complex protein, gp40 was localized to the nuclear rim, as shown by biochemical fractionation and immunofluorescence microscopy.
40 citations
TL;DR: Several components of the to the vacuole is mediated by short peptide sequences in secretory machinery have recently been identified in the protein or precursor proteins and are described in this review.
Abstract: and storage vacuoles is dependent upon specific targeting information. In mammalian systems, the targeting of Most proteins that are synthesized on membrane- soluble proteins to the lytic compartment, the lysosome, bound ribosomes are transported through the Golgi is frequently mediated by mannose 6-phosphate residues and reach the trans-Golgi network to be sorted for in glycosyl side-chains of the glycoproteins ( Kornfeld, delivery to various cellular destinations, including the 1990). Vacuolar acid hydrolases in plant systems are also vacuole. Sorting involves a recognition of proteins by glycoproteins (Gaudreault and Beevers, 1983); however, receptors and the assembly of cytosol-oriented coat plants do not contain mannose 6-phosphate residues or structures that package cargo into vesicles. Vesicle M-6-P receptors (Gaudreault and Beevers, 1984). Instead trafficking is regulated by specific membrane-bound the targeting of soluble proteins and some storage proteins and soluble proteins. Several components of the to the vacuole is mediated by short peptide sequences in secretory machinery have recently been identified in the protein or precursor proteins. These targeting signals plants and are described in this review. Ongoing and can occur at the amino terminus of propeptides (NTPP) future research will characterize features of the or at the carboxy terminus (CTPP), or they may be part secretory pathway specific to plants which, because of the mature protein (Chrispeels and Raikhel, 1992). of the multiplicity of vacuole types, provide a more The role of these peptide sequences is described in detail complex paradigm than the better described mamma- in the mini review by Nakamura and Neuhaus (1998). lian and yeast systems. In mammalian systems, the functioning of M-6-P as a targeting determinant is dependent upon the ability of
17 citations
01 Jan 1998
TL;DR: In eukaryotic cells, most proteins destined for the cell surface or for organelles of the secretory pathway are synthesized by endoplasmic-reticulum-bound ribosomes and co-translationally transported into the ER lumen.
Abstract: In eukaryotic cells, most proteins destined for the cell surface or for organelles of the secretory pathway are synthesized by endoplasmic-reticulum-bound ribosomes and co-translationally transported into the ER lumen. This translocation process is dependent on the presence of a signal sequence found at the N-terminus of most soluble secretory proteins. If no other signal is present, soluble proteins are transported to the cell surface by a default mechanism, with a positive sorting signal being required for retention within the endomembrane system (reviewed in Bednarek and Raikhel, 1992) [Fig. 1]. Soluble vacuolar proteins (in plant and yeast cells) or lysosomal proteins (in mammalian cells) are transported through the secretory pathway to the trans-Golgi network (TGN), where they are segregated from secreted proteins and carried to the vacuole or lysosome in clathrin-coated vesicles. The mechanisms for sorting proteins to these organelles differ between organisms. Sorting to mammalian lysosomes is usually mediated by oligosaccharide modifications, whereas sorting to yeast and plant vacuoles is mediated by short peptide domains that may be removed before or upon deposition in the organelle, or it may form a part of the mature protein (for review see, Bar-Peled et al., 1996). Soluble proteins are transported through the secretory system via transport vesicles that bud from one compartment and fuse specifically with the next. The specificity of these fusion events is thought to be determined by integral membrane proteins on the vesicles and the acceptor membrane and several candidates for these targeting molecules have recently been identified (Rothman, 1994).