Transport of proteins into chloroplasts. Binding of nuclear-coded chloroplast proteins to the chloroplast envelope

A system has been constructed in vitro for the binding of cytoplasmically synthesized chloroplast proteins to the chloroplast envelope which precedes the uptake into the organelle in vivo. Isolated chloroplast envelopes from young pea or spinach are capable of binding the majority of proteins obtained by translation of poly(A)-containing RNA from greening plants in vitro. Among the bound proteins the precursors to the light-harvesting chlorophyll a/b apoprotein and the small subunit of ribulose-1,5-bisphosphate carboxylase are prominent. Binding is an intrinsic property of the envelope membrane and does not require energy in the form of ATP. Bound proteins remain on the surface of the envelope vesicles and can be digested by protease. Binding is complete within minutes, shows a high affinity of the reactants, and is non-ionic in nature. Protein binding is specific for translation products of poly(A)-containing RNA from greening plants. Precursors to chloroplast protein are bound preferentially as compared to the mature proteins. The specificity is further demonstrated by the low binding of proteins obtained by run-off translation of polysomes. Binding of radioactive labeled proteins is subject to competition by excess unlabeled homologous proteins. Once bound, the proteins are withdrawn from competition indicating a high binding stability. All the properties found for binding of proteins to isolated envelopes are consistent with the concept of the so-called envelope carrier hypothesis.     

Targeting of proteins into chloroplasts

Most chloroplastic proteins are synthesized in the cytoplasm and are transported to their proper location as a posttranslational event. In the present paper we briefly review some aspects of this transport process. Because chloroplasts contain six different locations, one interesting aspect of protein targeting into chloroplasts that we consider is how precursor proteins are targeted to these various locations. One step shared by many proteins is transport across the envelope membranes. Although this process has been well studied, the components of the apparatus that mediate this transport step are mostly unidentified. Strategies to identify components of this transport apparatus are considered.     

Targeting of proteins into chloroplasts

Cytoplasmically synthesized proteins are directed into chloroplasts by amino terminal transit sequences of the precursor proteins. For proteins of the thylakoid lumen, transit sequences are also important in directing proteins to the lumen.     

Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol and targeted into the thylakoid lumen by bipartite presequences. In this report, we describe conditions for the efficient import of each of these proteins by isolated pea thylakoids. Import of the 33-kDa protein requires both light and stromal extract. The probable function of the stromal extract is to provide stromal processing peptidase to remove the first "envelope transit" signal of the presequence. Import of the 23-kDa protein is also driven by light, but stromal extract is not required for import; furthermore, efficient import is still observed if the precursor is modified to completely block cleavage by residual stromal processing peptidase activity. The intermediate form of the 23-kDa protein, which is generated by incubation of the precursor protein with stromal processing peptidase, is also efficiently imported. The results indicate that the thylakoidal protein transport system can import both the precursor and intermediate forms of the 23-kDa protein, but probably only the intermediate form of the 33-kDa protein.     

Transport of glutamine into isolated pea chloroplasts

Abstract. Uptake of [¹⁴C] glutamine into isolated pea chloroplasts has been examined by using a centrifugal filtration technique. Competition experiments showed that glutamine uptake is mediated by a dicarboxylate carrier with K_m 1.10 mM and V _max. 118 nmol of glutamine min⁻¹ per mg of chlorophyll. Isolated pea chloroplasts accumulated glutamine in the sucrose-impermeable space to concentrations higher than that present in the external solution when the latter was below 0.5 mM. It is suggested that glutamine accumulation is driven by exchange (utilizing the dicarboxylate carrier) with the endogenous pool of dicarboxylates in the chloroplasts. Increasing pH stimulated glutamine uptake but inhibited that of glutamate and 2-oxoglu-tarate. The hypothesis is advanced that when molecules of different charge are exchanged across the chloroplast envelope via the dicarboxylate carrier, electroneutrality is maintained by transport of protons, and that this explains the observed effects of increasing pH. The low rates of glutamine transport coupled with the strong competition of other dicarboxylates for the carrier suggest that export in vivo from the chloroplast of nitrogen in the form of glutamine is not of major importance.     

Import of proteins into mitochondria and chloroplasts

Although mitochondria and chloroplasts synthesize some of their own proteins, they must import most of them from the cytosol. Import is mediated by molecular chaperones in the cytosol, receptors and channels in the organelle membranes and ATP-driven 'import motors' inside the organelles. Many of these components are now known, allowing informed guesses on how they might work.     

Protein import into chloroplasts involves redox-regulated proteins

Pre-protein translocation into chloroplasts is accomplished by two distinct translocation machineries in the outer and inner envelope, respectively. We have isolated the translocon at the inner envelope membrane (Tic complex) by blue-native PAGE and describe a new Tic subunit, Tic62. Tic62, together with Tic110 and Tic55, forms a core translocation unit. The N-terminus of Tic62 shows strong homologies to NAD(H) dehydrogenases in eukaryotes and to Ycf39-like proteins present in cyanobacteria and non-green algae. The stromal-facing C-terminus of Tic62 contains a novel, repetitive module that interacts with a ferredoxin-NAD(P)(+) oxidoreductase. Ferredoxin-NAD(P)(+) oxidoreductase catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P). Substrates that interfere with either NAD binding, such as deamino-NAD, or influence the ratio of NAD(P)/NAD(P)H, such as ruthenium hexamine trichloride, modulate the import characteristics of leaf-specific ferredoxin-NAD(P)(+) oxidoreductase isologues differently. We conclude that the Tic complex can regulate protein import into chloroplasts by sensing and reacting to the redox state of the organelle.     

Transport of purines and pyrimidines into isolated pea chloroplasts

Abstract. Uptake of [¹⁴C] adenine and [¹⁴C] cytosine into isolated pea chloroplasts has been examined by using a centrifugal-filtration technique. These chloroplasts accumulate both compounds in the sucrose-impermeable space to concentrations higher than that present in the external solution when the latter was below 1.0 mM. Competition experiments suggested that uptake is mediated by a carrier mechanism.     

Transport of proteins into chloroplasts. Partial purification of a thylakoidal processing peptidase involved in plastocyanin biogenesis

Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported across three membranes into the chloroplast thylakoid lumen. Processing to the mature size involves successive cleavages by a stromal and a thylakoidal peptidase. In this report we describe the partial purification and characterization of the thylakoidal peptidase involved. The enzyme has been purified 36-fold from Pisum sativum thylakoids after solubilization using Triton X-100. The peptidase processes the plastocyanin import intermediate to the mature size, but no further, and is capable of processing pre-plastocyanin to the mature size but at a lower rate. No detectable activity is displayed against non-chloroplast proteins or precursors of stromal proteins. The enzyme has a pH optimum of 6.5-7 and is activated by chelating agents such as EDTA and EGTA. No inhibitors of the peptidase have been found to date.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

Travelling of proteins through membranes: translocation into chloroplasts

 Most proteins involved in plastid biogenesis are encoded by the nuclear genome. They are synthesised in the cytosol and have to be transported toward and subsequently translocated into the organelle. This targeting and import process is initiated by a specific chloroplast-targeting signal. The targeting signal of the preprotein is recognised and modified by cytosolic proteins which function in transport toward the chloroplast and in maintaining the import-competent state of the preprotein. The precursor is transferred onto a multi-component complex in the outer envelope of the chloroplasts, which is formed by receptor proteins and the translocation channel. Some proteins, not containing transit sequences, are directly sorted into the outer membrane whereas the majority, containing transit sequences, will be translocated into the stroma. This involves the joint action of a protein complex in the outer envelope, one complex in the inner envelope, and soluble proteins in the intermembrane space and the stroma. The origin of this translocation complex following the endosymbiotic events is an unsolved question. Recent identification of homologous proteins to some members of this machinery in the cyanobacterium Synechocystis PCC6803 gives an initial insight into the origin of the translocation complex. Received: 27 December 1999 / Accepted: 29 March 2000     

Transport of proteins into cryptomonads complex plastids

Complex plastids, found in many alga groups, are surrounded by three or four membranes. Therefore, proteins of the complex plastids, which are encoded in the cell nucleus, must cross three or four membranes during transport to the plastid. To study this process we have developed a method for isolating transport-competent two membrane-bound plastids derived from the complex plastids of the cryptophyte Guillardia theta. This in vitro protein import system provides the first non-heterologous system for studying the import of proteins into four-membrane complex plastids. We use our import system as well as canine microsomes to demonstrate in the case of cryptomonads how nuclear proteins pass the first nucleomorph-encoded proteins the third and fourth membrane and discuss the potential mechanisms for protein transport across the second membrane.     

Transport of nuclear-encoded proteins into secondarily evolved plastids

Many algal groups evolved by engulfment and intracellular reduction of a eukaryotic phototroph within a heterotrophic cell. Via this process, so-called secondary plastids evolved, surrounded by three or four membranes. In these organisms most of the genetic material encoding plastid functions is localized in the cell nucleus, with the result that many proteins have to pass three, four, or even five membranes to reach their final destination within the plastid. In this article, we review recent models and findings that help to explain important cellular mechanisms involved in the complex process of protein transport into secondary plastids.     

Transport of proteins into chloroplasts. Organization, orientation, and lateral distribution of the plastocyanin processing peptidase in the thylakoid network

Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen of the chloroplast. Maturation of preplastocyanin involves successive cleavages by a stromal peptidase and a distinct thylakoidal peptidase. In this report we have analyzed the precise location and orientation of the thylakoidal peptidase with respect to the thylakoid membrane. Experiments involving differential centrifugation of thylakoid extracts and sonication of isolated vesicles indicate that the peptidase is tightly bound to the thylakoid membrane but not intimately associated with any of the major thylakoid protein complexes. Analysis of the lateral distribution of the peptidase has shown that the enzyme is exclusively located in the non-appressed lamellae of the thylakoid network. The active site of the peptidase is on the lumenal face of the thylakoid membrane.