Chloroplast coupling factor 1: A species-specific receptor for tentoxin

Abstract

Produced by the phytopathogenic fungus Alternaria tenuis, it induces chlorosis in many plants, including lettuce, potato, cucumber, and spinach, but has little or no effect on others such as radish, tobacco, and corn (4, 5). The chlorosis results from a selective disruption of chloroplast function in that the amounts of lipids (6) and proteins (7) specific to chloroplasts are reduced and the ultrastructural alterations found are confined to the chloroplast (8). The sharp demarcation between sensitive and insensitive species suggested that chloroplasts from sensitive species might possess a specific receptor site for tentoxin. Because preliminary studies have shown that tentoxin inhibits phosphorylating electron transport in lettuce chloroplasts (9) and chloroplast coupling factor 1 (CF1) is directly associated with photophosphorylation (10), we investigated it as a potential receptor molecule for tentoxin. This paper presents evidence that CF1 from lettuce, a tentoxin-sensitive species, has a single binding site for tentoxin and that when tentoxin occupies this site, both CF1 ATPase and phosphorylating electron transport are inactivated. In contrast, CF1 from radish, an insensitive species, has a lower affinity for tentoxin than does lettuce CF1; tentoxin does not inhibit radish ATPase and only slightly inhibits photophosphorylation.MATERIALS AND METHODS Chloroplasts were prepared from leaves of lettuce (cv. Romaine) and spinach purchased locally and from seedlings of radish (cv. Comet) and other species grown in controlled environment chambers. One hundred grams of selected leaves were blended for 10 sec at 40 in 250 ml of a 0.4 M sucrose 0.05 M N-[tris(hydroxymethyl)methyl]glycine (Tricine) buffer, pH 8.0, (ST). The homogenate was filtered through eight layers of cheesecloth, and the chloroplasts were collected by centrifugation (1000 X g for 10 min). After washing in 40 ml of ST and an additional centrifugation, the chloroplast pellet was suspended in 10 ml of ST and adjusted to 1 mg of chlorophyll per ml (11).Electron transport was measured with an oxygen electrode using methods modified from those of Arntzen (9). Chloroplasts (35 ,g of chlorophyll) in ST were added to 3 ml of stirred reaction mixture at 280 followed by the addition of tentoxin. Next, the electrode was inserted during a brief cessation of stirring. Two minutes after the addition of tentoxin, the reaction chamber was illuminated with a tungsten lamp (15 klm/m2). The course of oxygen uptake was followed during the subsequent 1 min. Basal rates were measured in the presence of 3 mM ADP; complete rates were determined in the added presence of 5 mM NaH2PO4. Only preparations having a complete rate more than twice the basal rate were used.Coupling factor 1 was prepared from chloroplasts by the methods of Lien and Racker (12). Lettuce CF1 was judged to be pure by gel electrophoresis (13, 14) after chromatography on DEAE-Sephadex A-50 using a linear (NH4)2SO4 gradient (Step 3 of ref. 12). Radish CF1 of comparable purity was obtained after initial co...

Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

“…The Mg-ATPase activity of the isolated CF 1 containing the Asp residue in position 83 (␤E83D) was indeed inhibited by tentoxin at concentrations similar to those required to inhibit enzymes from sensitive plants and from the sensitive cyanobacterium Anacystis nidulans (Fig. 4B) (7,8,25). Effective inhibition by tentoxin required preincubation of the enzyme with tentoxin as reported previously (Fig.…”

“…Tentoxin, a cyclic tetrapeptide (cyclo-L-leucyl-N-methyl-(z)-dehydrophenylalanyl-glycyl-N-methyl-alanyl) produced by the fungus Alternaria tenuis, is a potent inhibitor of the chloroplast ATP synthase of certain sensitive plant species (8,9), which acts as an uncompetitive inhibitor of the CF 1 -ATPase with respect to nucleotide substrates. It was proposed that 1 mol of tentoxin/mol of CF 1 , bound at a site near the interface between the N-terminal domains of subunits ␣ and ␤, interferes with the cooperative interaction between nucleotide binding sites inhibiting the enzyme activity (10).…”

Proton coupling is preserved in membrane‐bound chloroplast ATPase activated by high concentrations of tentoxin

“…Its main target is the F~ moiety of some photosynthetic H+-ATPases [2][3][4]. The site of inhibition seems to be the fl subunit [5], although the ~ subunit is probably required for TTX binding [6,7].…”

Tentoxin‐induced adenine nucleotide exchange with soluble and thylakoid membrane‐bound chloroplast coupling factor 1

“…CF1 [16], tentoxin [17] and chloroplasts [3] were prepared as described. The specific activity of the protein used for this investigation was 23 #mol ATP hydrolyzed/mg protein/min, assayed as the Ca 2+-dependent ATPase after trypsin activation [18].…”