A chloroplast processing enzyme functions as the general stromal processing peptidase

Abstract

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.

Figure 1

Structure of CPE and expression of CPE-B fusion protein. (A) Alignment of N-terminal amino acid sequences of A. thaliana (A.t.) and P. sativum (P.s.) preCPE. Lowercase letters indicate different amino acids. Uppercase letters indicate amino acids with a homology index ≥0.8 (28). Dots represent identical amino acids. Sequence of mature CPE predicted from the alignment is indicated by shading. The zinc binding motif is underlined. (B) Schematic map defined by amino acid comparison of pea and Arabidopsis CPE. The highly conserved regions I to IV, the zinc-binding motif of conserved region I, and a putative flexible linker are indicated. To express active CPE, the predicted transit peptide was replaced by a biotin-containing peptide (CPE-B). (C) Western blot analysis of CPE-B expression in E. coli using the anti-145/143-kDa antibody was carried out as described (7). Soluble cell extracts of induced cells carrying pBEX5BA (lane 1), and of uninduced and induced cells carrying pCPE-B (lanes 2 and 3, respectively). CPE-B was partially purified with avidin-resin demonstrating biotinylation of the fusion protein (lane 4). Soluble chloroplast extract containing CPE (lane 5) was prepared as described (3).

Figure 2

CPE is the general SPP of chloroplasts. (A) Processing of [³⁵S]methionine-labeled preLHCPII wild type (wt) and a processing site mutant. (B) Processing of [³⁵S]methionine-labeled preRBCS, preRBCA, preCF₁γ, preOEE1, prePC, preACP1, preDHS1, and preHSP21. (C) Processing of [³⁵S]methionine-labeled preCPE. (D) Processing of preFD labeled with [³⁵S]methionine (Met*) and [³⁵S]cysteine (Cys*), respectively. Lanes 1, radiolabeled precursor. Lanes 2, precursor incubated with control extract of induced E. coli cells carrying pBEX5BA lacking the CPE insert, except incubation of preACP was done with magnetic beads that were preincubated with the control extract. Lanes 3, processing with CPE-B extract, except processing of preACP was carried out with immobilized CPE-B due to a proteolytic activity in the E. coli extract that degraded preACP. Lanes 4, processing with chloroplast extract. p, Precursor; i, intermediate; m, mature; tp, transit peptide.

Figure 3

Amino acid sequences of identified CPE cleavage sites. The processing sites of preRBCA (Spinacia oleracea)(10), preLHCPI (Lycopersicon esculentum)(43), preLHCPII (Triticum aestivum)(15), preOEE1 (T. aestivum)(44), preOEE2 (T. aestivum)(45) and prePC (Silene pratensis)(19) are shown. The amino acids found by N-terminal sequencing of the cleavage product of preRBCA generated by immobilized CPE-B are underlined. The length of the precursors is noted in parentheses. Position of the C-terminal amino acids of the transit peptides is shown. The cleavage sites are indicated by arrows.

Figure 4

CPE is a metal-dependent enzyme. (A) Percent of cleavage of [³⁵S]methionine-labeled preRBCA in the presence of EDTA (▪), EGTA (○), and 1,10-phenanthroline (•). Aliquots of CPE-B extract were preincubated at 28°C for 30 min with each inhibitor and activity was assayed under standard conditions with 1 μl of in vitro synthesized preRBCA. Processing products were separated by SDS/PAGE and quantified using PhosphorImager (Molecular Dynamics). Extent of cleavage in absence of inhibitor was taken as 100%. (B) Reactivation of immobilized CPE-B by Zn²⁺ in chelating buffer. Untreated [³⁵S]methionine-labeled preRBCA (lane 1), preRBCA incubated with immobilized CPE-B without inhibitor preincubation (lane 2), and after preincubation with 1 mM 1,10-phenanthroline at 0 μM (lane 3), 5 μM (lane 4), and 50 μM (lane 5) ZnCl₂.