Three proteins mediate import of transit sequence-less precursors into the inner envelope of chloroplasts in Arabidopsis thaliana

Abstract

A family of 17 putative preprotein and amino acid transporters designated PRAT has been identified in Arabidopsis thaliana, comprising PRAT proteins in mitochondria and chloroplasts. Although some PRAT proteins, such as the translocon of the mitochondrial inner membrane (TIM) proteins TIM22 and TIM23, play decisive roles for the translocation and import of mitochondrial inner membrane proteins, little is known about the role of the different PRAT members in chloroplasts. Here we report the identification of three distinct PRAT proteins as part of a unique protein import site. One of the identified PRAT proteins is identical with a previously characterized hypothetical protein (HP) of 20 kDa designated HP20 of the outer plastid envelope membrane. The second PRAT component is represented by HP30, and the third is identical to HP30-2, a close relative of HP30. Both HP30 and HP30-2 are inner plastid envelope membrane proteins of chloroplasts. Using biochemical, cell biological, and genetic approaches we demonstrate that all three PRAT proteins cooperate during import of transit sequence-less proteins, such as the quinone oxidoreductase homolog ceQORH used as model, into the inner chloroplast envelope membrane. Our data are reminiscent of findings reported for the TIM22 translocase, which is involved in the import of carrier proteins and other, hydrophobic membrane proteins lacking cleavable transit sequences into the inner mitochondrial membrane. Together our results establish the PRAT family as a widely used system of protein translocases in different membranes of endosymbiotic origin.chloroplast biogenesis | membrane transport | protein translocation P lastids represent a highly divergent family of cell organelles (1). They are ubiquitously found in plant and algal cells and provide essential metabolic and signaling functions (2, 3). The hallmark organelles of green plants are chloroplasts that contain the green pigment chlorophyll and perform photosynthesis for autotrophic growth (1-3).Chloroplasts contain ∼3,000 proteins, of which most are nuclear gene products (4). The majority of these proteins is synthesized with NH 2 -terminal, cleavable extensions referred to as chloroplast transit peptides, which guide the cytosolic precursors to the outer plastid envelope membrane and initiate membrane translocation through two closely interacting multiprotein complexes, called translocon of the outer chloroplast envelope (TOC) and translocon of the inner chloroplast envelope (TIC) (5). During or shortly after membrane passage, the NH 2 -terminal transit peptides are cleaved off proteolytically (5).Proteomics studies by Kleffmann et al. (6) revealed a set of plastid proteins lacking cleavable transit peptides. Therefore, the existence of alternative targeting signals and import pathways was proposed (6). Examples for inner envelope membrane proteins that lack a cleavable transit peptide are TIC32 (7) and a protein homologous to quinone oxidoreductases of bacteria, yeast, and animals, designated chloroplast-envelope quinon...

New functions of the chloroplast Preprotein and Amino acid Transporter (PRAT) family members in protein import

“…9 These results imply initial interactions of ceQORH in the outer chloroplast envelope with HP20 and late interactions of ceQORH in the inner chloroplast envelope with HP30/HP30-2. Second, protease protection assays revealed different susceptibilities of HP20 and HP30 to thermolysin and trypsin 9 that are explained only if HP20 and HP30 have different locations in chloroplasts. While HP20 was sensitive to both treatments, HP30 was insensitive to thermolysin and partially susceptible to added trypsin.…”

“…14 In our recent paper by Rossig et al 9 we now identified this and 2 new crosslink products and show that all 3 proteins belong to the PRAT family comprising preprotein and amino acid transporters in mitochondria and chloroplasts. 15,16 These cross-linked proteins were originally identified by proteomics studies on isolated envelopes as hypothetical proteins 17,18 and defined by their apparent molecular mass, giving rise to HP20, HP30, and HP30-2.…”

“…19,20 Third, fractionation experiments demonstrated the presence of HP20 vs. HP30/HP30-2 in outer and inner chloroplast envelope membranes, respectively. 9 Whereas HP20 co-fractionated with outer envelope marker proteins such as TOC75, HP30 and HP30-2 co-fractionated with inner envelope marker proteins such as TIC110. 9 Carbonate and salt extraction experiments revealed that all 3 HP proteins represent integral membrane proteins.…”

“…For example, it has been used to analyze bacterial protein translocation and probe the interplay of the protein-conducting channel formed by the SecY complex and the cytoplasmic SecA ATPase. 10 In the work recently published by Rossig et al, 9 disulfide bridge cross-linking with Elman's reagent (5,5'-dithiobis(2-nitrobenzoic acid, DTNB; Fig. 1) was used to identify components that mediate the import of transit sequence-less proteins into the inner envelope of chloroplasts.…”

“…While HP20 was sensitive to both treatments, HP30 was insensitive to thermolysin and partially susceptible to added trypsin. 9 Thermolysin is a protease which degrades only surface-exposed plastid proteins, whereas trypsin penetrates the outer envelope membrane and also degrades inner envelope membrane proteins up to their membrane parts. 19,20 Third, fractionation experiments demonstrated the presence of HP20 vs. HP30/HP30-2 in outer and inner chloroplast envelope membranes, respectively.…”