Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-␣-glucanotransferase (EC 2.4.1.25) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of ϳ150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the -anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.During photosynthesis, as much as one-half of the carbon fixed during the day is stored as transitory starch inside chloroplasts for remobilization at night. The pathway by which starch in leaves is converted to sucrose has only recently been elucidated (1) and some questions remain. The breakdown of storage starch upon seed germination takes place essentially outside of cells (cellular integrity is lost) while transitory starch must be broken down in intact chloroplasts and cells (2). Transitory starch must have some phosphate attached in order to be remobilized at night (3-6). During the day, carbon is exported from the chloroplast almost exclusively as triose phosphate, but at night maltose is exported (7,8). Starch is acted on by -amylase (9), and the resulting -maltose is exported from chloroplasts through a novel maltose transporter (10). Plants lacking this transporter accumulate maltose in their plastids (11). In the cytosol, maltose metabolism requires a 4-␣-glucanotransferase (EC 2.4.1.25). This enzyme was called the cytosolic D enzyme and, by analogy with the plastidial D enzyme, was believed to have no activity with maltose (12). Arabidopsis thaliana plants lacking this enzyme accumulate up to 100-fold more maltose than wild type and grow significantly more slowly (13,14). Potato plants in which this enzyme was reduced in activity accumulated high levels of maltose in leaves but leaf starch synthesis and starch metabolism in tubers was not affected (15).This enzyme is now called disproportionating enzyme 2 (DPE2) 3 because of its similarity to the disproportionat...
The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection.
The leader (L) protein of encephalomyocarditis virus (EMCV) shuts off host cell nucleocytoplasmic trafficking (NCT) by inducing hyperphosphorylation of nuclear pore proteins. This dramatic effect by a nonenzymatic protein of 6 kDa is not well understood but clearly involves L binding to cellular Ran GTPase, a critical factor of active NCT. Exogenous GDP and GTP are inhibitory to L-Ran binding, but the guanine-nucleotide exchange factor RCC1 can relieve this inhibition. In the presence of RCC1, L binds Ran with a K D (equilibrium dissociation constant) of ϳ3 nM and reaches saturation within 20 min. The results of fluorescently tagged nucleotide experiments suggest that L-Ran interactions affect the nucleotide-binding pocket of Ran. E ncephalomyocarditis virus (EMCV) infection shuts down active nucleocytoplasmic trafficking (NCT) by inducing the phosphorylation of PheGly-containing nuclear pore proteins (Nups), potentially blocking otherwise toxic antiviral responses (1-4). The leader (L) protein of EMCV alone is responsible for this activity. In the context of cytoplasm and nuclear pore targets, L induces irreversible NCT inhibition, new cellular mRNA transcripts are unable to be exported from the nucleus, and nucleartargeted cytoplasmic proteins fail to be actively imported (3,5,6).L is a small protein (67 amino acids) with a novel N-terminal CHCC zinc-finger motif and a highly acidic (pI 3.8) -hairpin motif (7,8). Despite being sufficient to shut down NCT, L itself has no predicted or observable enzymatic activity. It must act by binding or abrogating cellular and/or viral cofactors to bring about such significant cellular responses (9). Indeed, among the known binding partners of L is Ran GTPase, a central component of all active NCT (10).Previous studies have shown that L not only interacts with Ran, it also inhibits critical events that are dependent on Ran GTP/GDP cycling (3, 10). To understand how L redirects Ran's role and triggers consequent Nup phosphorylation is of interest, not only of GDP or GTP (2.5 M) and/or RCC1-GST (1 nM). The clarified supernatant (Unbound) and bead-bound proteins (Bound) were precipitated (30% trichloroacetic acid), solubilized (alkaline SDS), fractionated by PAGE, and then transferred to polyvinylidene difluoride membranes. Western blot analyses used anti-Ran polyclonal antibody (product no. SC-1156; Santa Cruz Biotech) or anti-GST monoclonal antibody (product no. 71097; Novagen). Secondary antibodies were horseradish peroxidase-conjugated anti-goat antibody (product no. A5420; Sigma) or anti-mouse antibody (product no. A2554; Sigma). Relative pixel counts (% total) were determined by ImageQuant (GE Life Sciences) scanning of the membranes. to the field of virus-host interactions but also to the field of mitotic molecular biology, in which Ran serves other central functions. (10) were expressed and purified from Escherichia coli. GST-tagged RCC1 was generated from pGEX-RCC1 (a kind gift from C. Wiese). Previous data have shown that GST-L can pull down native Ran from HeLa cel...
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