BBA - Molecular Cell Research (v.1541, #1-2)

Preface (1).

Transit peptides are N-terminal extensions that facilitate the targeting and translocation of cytosolically synthesized precursors into plastids via a post-translational mechanism. With the complete Arabidopsis genome in hand, it is now evident that transit peptides direct more than 3500 different proteins into the plastid during the life of a typical plant. Deciphering a common mechanism for how this multitude of targeting sequences function has been hampered by the realization that at a primary sequence level, transit peptides are highly divergent in length, composition, and organization. This review addresses recent findings on several of the diverse functions that transit peptides must perform, including direct interaction with envelope lipids, association with a cis-acting guidance complex, recognition by envelope receptors, insertion into the Toc/Tic translocon, interaction with molecular motors, and finally, recognition/cleavage by the stromal processing peptidase. In addition to higher plants, transit peptides also direct the import of proteins into complex plastids derived from secondary endosymbiosis. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or possibly overlapping functions. Although still poorly characterized, evolutionary processes could yield transit peptides with alternative domain organizations.
Keywords: Organelle biogenesis; Protein transport; Endosymbiosis; GTPase; Envelope; Galactolipid;

The chloroplast membranes are highly regulated and biological active regions of the living plant cell, which carry numerous essential proteinaceous components. For example, in the thylakoid membrane the photosynthesis apparatus, one of the most life-relevant biological machineries, is located. How these membrane proteins are targeted to and inserted into their target membranes was one of the questions we aimed to understand in the last few years. Fifteen years ago little to nothing was known about the targeting and translocation of outer envelope proteins (G.W. Schmidt and L.M. Mishkind, Annu. Rev. Biochem. 55 (1986)). Although several protein assisted pathways for translocation of proteins across the membranes have been characterised, only recent results gave insight into how membrane proteins are inserted into the chloroplast membranes. Here we will focus on the mode of insertion of a class of proteins into the outer envelope and the thylakoid membranes, which share a unique feature: they insert apparently directly into the lipid bilayer, i.e. without the help of a proteinaceous translocation pore.
Keywords: Outer envelope; Thylakoid; Membrane insertion; Direct insertion; Targeting;

Translocation of proteins across the multiple membranes of complex plastids by Giel G van Dooren; Steven D Schwartzbach; Tetsuaki Osafune; Geoffrey I McFadden (34-53).
Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted.
Keywords: Malaria; Protein transport; Endoplasmic reticulum; Vesicle; Golgi apparatus;

Dual targeting to mitochondria and chloroplasts by Nemo Peeters; Ian Small (54-63).
Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.
Keywords: Mitochondria; Chloroplast; Protein import; Dual targeting;

Toc, Tic, and chloroplast protein import by Paul Jarvis; Jürgen Soll (64-79).
The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex – comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components – docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc ( t ranslocon at the o uter envelope membrane of c hloroplasts) and Tic ( t ranslocon at the i nner envelope membrane of c hloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today’s import machinery was built around a prokaryotic core.
Keywords: Chloroplast protein import; Translocon; Toc apparatus; Tic apparatus; Chloroplast envelope; Protein targeting;

Two distinct protein translocation pathways that employ hydrophobic signal peptides function in the plant thylakoid membrane. These two systems are precursor specific and distinguished by their energy and component requirements. Recent studies have shown that one pathway is homologous to the bacterial general export system called Sec. The other one, called the ΔpH-dependent pathway, was originally considered to be unique to plant thylakoids. However, it is now known that homologous transport systems are widely present in prokaryotes and even present in archaea. Here we review these protein transport pathways and discuss their capabilities and mechanisms of operation.
Keywords: Tat; Twin arginine; Folded protein transport; Chloroplast; Amphipathic helix; Signal peptide;

Biogenesis and origin of thylakoid membranes by Ute C. Vothknecht; Peter Westhoff (91-101).
Thylakoids are photosynthetically active membranes found in Cyanobacteria and chloroplasts. It is likely that they originated in photosynthetic bacteria, probably in close connection to the occurrence of photosystem II and oxygenic photosynthesis. In higher plants, chloroplasts develop from undifferentiated proplastids. These contain very few internal membranes and the whole thylakoid membrane system is built when chloroplast differentiation takes place. During cell and organelle division a constant synthesis of new thylakoid membrane material is required. Also, rapid adaptation to changes in light conditions and long term adaptation to a number of environmental factors are accomplished by changes in the lipid and protein content of the thylakoids. Thus regulation of synthesis and assembly of all these elements is required to ensure optimal function of these membranes.
Keywords: Chloroplast; Photosynthesis; Plastid development; Organelle evolution; Thylakoid formation;

Molecular chaperones involved in chloroplast protein import by Diane Jackson-Constan; Mitsuru Akita; Kenneth Keegstra (102-113).
Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address.
Keywords: Chloroplast; Protein targeting; Precursor protein; Molecular chaperone; Heat shock protein;

Prediction of organellar targeting signals by Olof Emanuelsson; Gunnar von Heijne (114-119).
The subcellular location of a protein is an important characteristic with functional implications, and hence the problem of predicting subcellular localization from the amino acid sequence has received a fair amount of attention from the bioinformatics community. This review attempts to summarize the present state of the art in the field.
Keywords: Chloroplast; Mitochondria; Presequence; Protein import; Sorting;

Function of a chloroplast SRP in thylakoid protein export by L.A. Eichacker; R. Henry (120-134).
Protein export systems derived from prokaryotes are used to transport proteins into or across the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane. Signal recognition particle (SRP) and its receptor are essential components used exclusively for cotranslational export of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and export of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes. An organellar SRP in chloroplasts (cpSRP) participates in cotranslational targeting of chloroplast synthesized integral thylakoid proteins. Remarkably, cpSRP is also used to posttranslationally localize a subset of nuclear encoded thylakoid proteins. Recent work has begun to reveal the basis for cpSRP’s unique ability to function in co- and posttranslational protein localization, yet much is left to question. This review will attempt to highlight these advances and will also focus on the role of other soluble and membrane components that are part of this novel organellar SRP targeting pathway.
Keywords: Signal recognition particle; Signal recognition particle; Oxa1; Alb3; Protein transport; FtsY; Thylakoid; Chloroplast;