Proteolytic Cleavage within a Regulatory Region of the γ Subunit of Chloroplast Coupling Factor 1

Abstract

The gamma subunit of chloroplast coupling factor 1 (CF1) is susceptible to selective proteolysis when the enzyme is in solution and the epsilon subunit is removed [CF1(-epsilon)]. In spinach thylakoid membranes, rapid cleavage of gamma is dependent on the generation of an electrochemical proton potential. The tryptic cleavage sites within the gamma of oxidized CF1 in illuminated thylakoids as well as of reduced CF1(-epsilon) in solution were determined by N-terminal amino acid sequencing. Two large gamma fragments of 27 000 (gamma27) and 10 000 (gammma10) molecular weight were generated by trypsin treatment of membrane-bound CF1. THe N-terminal gamma27 contains amino acids 1-215, and the C-terminal gamma10 contains 232-323. These polypeptides were tightly associated with the trypsin-resistant core of the enzyme. In contrast, three large gamma fragments were produced by trypsinolysis of reduced CF1(-epsilon). These polypeptides, which were also tightly associated with the trypsin-resistant core, have molecular weights of 7 900(gamma8), 14 850 (gamma15), and 10 000 (gamma10). These fragments contain residues 1-70, 71-204, and 232-323, respectively. The C-terminal gamma10 fragment generated by trypsin treatment of membrane-bound and soluble CF1 are identical. These results suggest that the gamma subunit of CF1 in illuminated thylakoids resembles that of CF1(-epsilon) with respect to accessibility to proteolytic cleavage. Cleavage of gamma between residues 215 and 232 is sufficient to fully activate the ATPase activity of the enzyme without reduction of the gamma disulfide. In addition, cutting within this region is responsible for loss of affinity for the inhibitory epsilon subunit.

Fragments of ATP synthase mediate plant perception of insect attack

“…The cATPC protein resides in CF 1 and contains an extra domain, absent from mitochondrial ATPC, spanning the inceptin peptide (Fig. 5A), which is susceptible to trypsin cleavage (27). ATPase is activated by reduction of the cATPC cysteine disulfide bridge, present in the inceptin fragment, which abolishes interactions with the inhibitory -subunit (27).…”

“…5A), which is susceptible to trypsin cleavage (27). ATPase is activated by reduction of the cATPC cysteine disulfide bridge, present in the inceptin fragment, which abolishes interactions with the inhibitory -subunit (27). Thus, inceptin fragments are derived from a regulatory domain unique to cATP synthases.…”

Cowpea Chloroplastic ATP Synthase Is the Source of Multiple Plant Defense Elicitors during Insect Herbivory

“…Symbols for treatments are denoted in the legend. et al, 1988;Schmelz et al, 2006) that is readily cleaved by trypsin (Hightower and McCarty, 1996), a predominant digestive enzyme in armyworm larvae (Paulillo et al, 2000). The E-inducing activity of OS collected at time zero from actively feeding larvae, bioassayed on cowpea leaves, was not significantly different between cowpea-and spinach-fed larvae (Fig.…”

“…The midguts of armyworm harbor potent trypsin and chymotrypsin endopeptidase activities; thus, Lys and Phe cleavage sites near the termini of the Gm-In ( 1 KGEICDVNGVCVDAAEDEF 2 ) are predicted to facilitate this process (Keil, 1992;Paulillo et al, 2000). Spinach cATPC harbors an additional trypsin cleavage site within the disulfide bridge of inceptin (Miki et al, 1988;Hightower and McCarty, 1996) and predictably results in larval OS devoid of cowpea E-inducing activity within 1 h (Fig. 6).…”

Photoinactivation of the F1-ATPase from Spinach Chloroplasts by Dequalinium Is Accompanied by Derivatization of Methionine β183

“…DTT treatment reduces the disulfide bond within the ␥ subunit (8), while heat activation relieves inhibition caused by the ⑀ subunit (9, 10). Trypsin digestion partially cleaves the ␥ subunit, which significantly decreases the affinity of CF 1 for the ⑀ subunit (11,12). The effects of octylglucoside and organic solvents on CF 1 are more complicated.…”