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...
Batch cultures of photoautotrophic cell suspensions of Chenopodium rubrum L., growing in an inorganic medium on CO2 under a daily balanced light-dark regime of 16: 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3-4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohormones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf.
Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3g49560 (HP30) and At5g24650 (HP30-2), in planta using a combination of genetic, cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria, whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes, one containing the inner membrane translocase TIM22 and the other containing an alternative NAD(P)H dehydrogenase subunit (NDC1) implicated in a respiratory complex 1-like electron transport chain. Through its association with TIM22, HP30-2 is involved in the uptake of carrier proteins and other, hydrophobic membrane proteins lacking cleavable NH -terminal presequences, whereas HP30-2's interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.
Common principles of protein translocation across membranesCells contain different, membrane-limited compartments endowed with specific proteins. To maintain these compartments, cells have developed elaborate systems for transporting the cell's few thousand different proteins to their final location. Export and import systems have been identified in bacteria and eukaryotes that operate by a similar set of principles.1 Each protein carries NH 2 -terminal or internal targeting information that is recognized by cytoplasmic targeting factors and directs the protein to specific receptors on the target membrane; the targeting signal interacts with a hetero-oligomeric trans-membrane channel that is gated both across and within the plane of the membrane. Molecular chaperones act as nucleoside triphosphate-powered import motors and interact with both the translocating polypeptide chain and the protein-conducting channel. Methods that have led to the identification of these principles include protein biochemistry, cell biology, and genetics.2-8 For example, it was found that mitochondria contain 2 types of receptors on their outer surface to bind cytosolic precursors for import. Mitochondrial precursors containing cleavable NH 2 -terminal pre-sequences interact with TOM20. By contrast, carrier proteins and other hydrophobic membrane proteins lacking cleavable NH 2 -terminal pre-sequences bind TOM70. A third TOM protein, TOM22, was found to associate with both TOM70 and TOM20. All 3 TOM proteins are essential for the formation of intact mitochondria and cooperate in the passage of precursors into the TOM40 import channel in the outer mitochondrial membrane.2-4 Import of pre-sequence-containing and pre-sequence-less precursors in fact converges at TOM40. 2-4Numerous other TOM proteins assist in subsequent steps of mitochondrial protein import. 2-4Import of pre-sequence-containing chloroplast precursors is mediated by at least 3 proteins: the 2 GTP binding receptors TOC159 and TOC34, and the β-barrel protein and translocation channel TOC75 5-8 . TOC159 and TOC34 are encoded by small gene families in Arabidopsis thaliana, with AtTOC159 containing 4 members, designated Attoc159, Attoc132, Attoc120, and Attoc90, and AtTOC34 containing 2 members, named Attoc34 and Attoc33. Biochemical and genetic evidence suggests that there is a functional specialization among the different members of the guanosine triphosphate hydrolyzing GTPase receptor families. 5-8Import of most pre-sequence-containing cytosolic precursors is Keywords: Membrane transport, Protein translocation, Chloroplasts and mitochondria, TIC and TIM translocons at the inner chloroplast envelope and inner mitochondrial membrane, TOC and TOM translocons at outer chloroplast and outer mitochondrial membrane, PRAT protein evolutionPlant cells contain distinct compartments such as the nucleus, the endomembrane system comprising the endoplasmic reticulum and Golgi apparatus, peroxisomes, vacuoles, as well as mitochondria and chloroplasts. All of these compartments are surroun...
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