sp1 cDNA was isolated from aspen (Populus tremula) plants by immunoscreening an expression library using polyclonal antibodies against BspA protein. BspA, which is a boiling-stable protein, accumulates in aspen plants in response to water stress and abscisic acid application (Pelah et al., 1995). The sp1 cDNA was found to encode a 12.4-kD generally hydrophilic protein with a hydrophobic C terminus, which is different from the BspA protein and was termed SP1 (stable protein 1). Northern-blot analysis revealed that sp1 encodes a small mRNA (about 0.6 kb) that is expressed in aspen plants under non-stress conditions and is accumulated after salt, cold, heat, and desiccation stress, and during the recovery from stress. The SP1 detected in plants remained soluble upon boiling, migrated both as a 12.4-kD band and a much higher mass of 116 kD on a 17% (w/v) Tricine-sodium dodecyl sulfate-polyacrylamide gel. Comparative protease digestion patterns, amino acid analyses, and the N-terminal sequences of the 12.4-and 116-kD proteins revealed that SP1 is homo-oligomeric. Furthermore, gel filtration chromatography analysis indicated that SP1 exists in aspen plants as a complex, composed of 12 subunits of 12.4 kD. A large number of sequences deduced from expressed sequence tags and genomic sequences of other organisms with unknown function show high homology to SP1. Thus, SP1 may represent a new protein family. Here, we present the first report on this putative protein family: the cloning, isolation, and characterization of SP1, a stress-responsive, boilingsoluble, oligomeric protein.It is well recognized that various environmental stresses significantly limit crop productivity. Drought, salinity, extreme temperatures, and oxidative stress, among others, are often interconnected and may induce similar cellular damage. For example, drought and salinization, sometimes in combination, are manifested primarily as osmotic stress, resulting in disrupted homeostasis of water potential and ion distribution in the cell (Serrano et al., 1999;Zhu, 2001a). Oxidative stress occurs frequently in the presence of high temperature, salinity, or drought stress, and causes the denaturation of functional and structural proteins (Smirnoff, 1998). As a consequence, these diverse environmental stresses often activate similar cell signaling pathways (Shinozaki and Yamaguchi-Shinozaki, 2000;Knight and Knight, 2001;Zhu, 2001b) and cellular responses, such as the production of stress proteins, up-regulation of antioxidants, and accumulation of compatible solutes (Vierling and Kimpel, 1992;Bray, 1993;Zhu et al., 1997;Cushman and Bohnert, 2000).To cope with environmental stresses, plants have developed various molecular-biochemical mechanisms that are involved in stress tolerance (Vierling, 1991;Ingram and Bartels, 1996;Bohnert and Sheveleva, 1998;Thomashow, 1999;Hoekstra et al., 2001). One of the mechanisms that may confer such tolerance is the activation of a large set of genes, leading to the accumulation of specific proteins in the cells. Late embryog...

Biochemical studies have identified two proteins, RB47 and RB60, that are involved in the light-regulated translation of the psbA mRNA in the chloroplast of the unicellular alga Chlamydomonas reinhardtii. RB47, a member of the eukaryotic poly(A)-binding protein family, binds directly to the 5' untranslated region of the mRNA, whereas RB60, a protein disulfide isomerase (PDI), is thought to bind to RB47 and to modulate its activity via redox and phosphorylation events. Our present studies show that RB47 forms a single disulfide bridge that most probably involves Cys143 and Cys259. We found that RB60 reacts with high selectivity with the disulfide of RB47, suggesting that the redox states of these two redox partners are coupled. Kinetics analysis indicated that RB47 contains two fast reacting cysteines, of which at least one is sensitive to changes in pH conditions. The results support the notion that light controls the redox regulation of RB47 function via the coupling of RB47 and RB60 redox states, and suggest that light-induced changes in stromal pH might contribute to the regulation.

Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteome ™ GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors cooperate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated.

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