Protein translocation across mitochondrial membranes.

Protein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes. In particular we will focus on recently identified components of the mitochondrial import apparatus.     

Protein translocation across chloroplast envelope membranes

Nuclear-encoded chloroplast proteins are imported from the cytosol into the chloroplast stroma by a common translocation machinery. Several components of the import apparatus, including GTP-binding proteins and Hsp70 proteins, have recently been identified and characterized. This review discusses the role of these proteins in chloroplast protein import.     

Protein translocation across and integration into membranes

This review concentrates mainly on the translocation of proteins across the endoplasmic reticulum membrane and cytoplasmic membrane in bacteria. It will start with a short historical review and will pinpoint the crucial questions in the field. Special emphasis will be given to the present knowledge on the molecular details of the first steps, i.e., on the function of the signal recognition particle and its receptor. The knowledge on the signal peptidase and the ribosome receptor(s) will also be summarized. The various models for the translocation of proteins across and the integration of proteins into membranes will be critically discussed. In particular, the function of signal, stop-transfer, and insertion sequences will be dealt with and molecular differences discussed. The cotranslational mode of membrane transfer will be compared with the post-translational transport found for mitochondria and chloroplasts. This review will conclude with open questions and an outlook.     

Protein translocation into and across the chloroplastic envelope membranes

Post-translational protein import into chloroplasts follows a common route characterised by the need for nucleoside-triphosphates at various steps and two distinct protein import machineries at the outer and inner envelope membrane, respectively. Several subunits of these complexes have been elucidated. In contrast, protein translocation into the chloroplastic outer envelope uses distinct and various but poorly characterised insertion pathways. A topological framework for single-membrane spanning proteins of the chloroplastic outer envelope is presented.     

Protein translocons: multifunctional mediators of protein translocation across membranes

Protein translocation systems consist of complex molecular machines whose activities are not limited to unidirectional protein targeting. Protein translocons and their associated receptor systems can be viewed as dynamic modular units whose interactions, and therefore functions, are regulated in response to specific signals. This flexibility allows translocons to interact with multiple signal receptor systems to manage the targeting of topologically distinct classes of proteins, to mediate targeting to different suborganellar compartments, and to respond to stress and developmental cues. Furthermore, the activities of translocons are tightly coordinated with downstream events, thereby providing a direct link between targeting and protein maturation.     

Protein translocation across biological membranes: cost what it may

Protein translocation across biological membranes can expend a large portion of the total cellular metabolic energy. Although energy sources for this process have been well characterized, its total energy cost was unclear. Recently, this problem was addressed in the thylakoidal pH-dependent cpTat pathway. It was estimated that approximately 3% of the total energy output of the chloroplast is used by this pathway.     

Protein translocation across membranes: common themes in divergent organisms

Specific signal sequences are required for the translocation of proteins into and across both the endoplasmic reticulum of eukaryotes and the plasma membrane of prokaryotes. The similar properties of these signals, together with their ability to function when transferred between systems, suggested that the mechanisms of translocation in the two cases may be fundamentally similar. Indeed, recent findings have revealed striking similarities between essential components of the prokaryotic and eukaryotic translocation systems, suggesting that both are derived from a common ancestor.     

Protein translocation across wheat germ microsomal membranes requires an SRP-like component

Different wheat germ extracts were tested for the presence of membranes capable of translocating and processing nascent secretory proteins. One lysate was found in which nascent prehuman-placental lactogen (phPL) was translocated and processed to mature human placental lactogen (hPL). Processing was found to occur concomitant with translocation across membranes. Translocation across the wheat germ membrane required a component which is similar to the mammalian signal recognition particle (SRP). It bound to DEAE–Sepharose, had a sedimentation coefficient of 11S and contained a 7S RNA. In addition to hPL, the plant protein zein and the bacterial protein β-lactamase were translocated across and processed by wheat germ membranes. Transport was found to occur only co-translationally. Our results show that the wheat germ protein translocation system is similar to the mammalian one. Unlike the mammalian SRP, the particle purified from wheat germ did not arrest elongation of nascent secretory proteins.     

Protein translocation across the peroxisomal membrane

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism, of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs, through a large membrane protein assembly.     

Posttranslational translocation of influenza virus hemagglutinin across microsomal membranes.

The biosynthesis of influenza virus hemagglutinin (HA) and its translocation across microsomal membranes were studied in a mammalian cell-free system. All forms of HA could be cotranslationally translocated with high efficiency. However, only truncated forms of HA were translocated after protein synthesis has been terminated. The efficiency of this posttranslational translocation was dependent on the extent of the truncation. Posttranslational translocation was ribosome dependent and occurred only in the presence of a functional N-terminal signal sequence. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed.     

Mechanism for translocation of fluoroquinolones across lipid membranes

Classical atom-scale molecular dynamics simulations, constrained free energy calculations, and quantum mechanical (QM) calculations are employed to study the diffusive translocation of ciprofloxacin (CPFX) across lipid membranes. CPFX is considered here as a representative of the fluoroquinolone antibiotics class. Neutral and zwitterionic CPFX coexist at physiological pH, with the latter being predominant. Simulations reveal that only the neutral form permeates the bilayer, and it does so through a novel mechanism that involves dissolution of concerted stacks of zwitterionic ciprofloxacins. Subsequent QM analysis of the observed molecular stacking shows the important role of partial charge neutralization in the stacks, highlighting how the zwitterionic form of the drug is neutralized for translocation. The findings propose a translocation mechanism in which zwitterionic CPFX molecules approach the membrane in stacks, but they diffuse through the membrane as neutral CPFX monomers due to intermolecular transfer of protons favored by partial solvation loss. The mechanism is expected to be of importance in the permeation and translocation of a variety of ampholitic drugs with stacking tendencies.     

Vesicle size-dependent translocation of penetratin analogs across lipid membranes

The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles (>1 microm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration.     

The very early evolution of protein translocation across membranes

In this study, we used a computational approach to investigate the early evolutionary history of a system of proteins that, together, embed and translocate other proteins across cell membranes. Cell membranes comprise the basis for cellularity, which is an ancient, fundamental organizing principle shared by all organisms and a key innovation in the evolution of life on Earth. Two related requirements for cellularity are that organisms are able to both embed proteins into membranes and translocate proteins across membranes. One system that accomplishes these tasks is the signal recognition particle (SRP) system, in which the core protein components are the paralogs, FtsY and Ffh. Complementary to the SRP system is the Sec translocation channel, in which the primary channel-forming protein is SecY. We performed phylogenetic analyses that strongly supported prior inferences that FtsY, Ffh, and SecY were all present by the time of the last universal common ancestor of life, the LUCA, and that the ancestor of FtsY and Ffh existed before the LUCA. Further, we combined ancestral sequence reconstruction and protein structure and function prediction to show that the LUCA had an SRP system and Sec translocation channel that were similar to those of extant organisms. We also show that the ancestor of Ffh and FtsY that predated the LUCA was more similar to FtsY than Ffh but could still have comprised a rudimentary protein translocation system on its own. Duplication of the ancestor of FtsY and Ffh facilitated the specialization of FtsY as a membrane bound receptor and Ffh as a cytoplasmic protein that could bind nascent proteins with specific membrane-targeting signal sequences. Finally, we analyzed amino acid frequencies in our ancestral sequence reconstructions to infer that the ancestral Ffh/FtsY protein likely arose prior to or just after the completion of the canonical genetic code. Taken together, our results offer a window into the very early evolutionary history of cellularity.     

Comparative and evolutionary aspects of macromolecular translocation across membranes

Membrane barriers preserve the integrity of organelles of eukaryotic cells, yet the genesis and ongoing functions of the same organelles requires that their limiting membranes allow import and export of selected macromolecules. Multiple distinct mechanisms are used for this purpose, only some of which have been traced to prokaryotes. Some can accomodate both monomeric and also large heterooligomeric cargos. The best characterized of these is nucleocytoplasmic transport. This synthesis compares the unidirectional and bidirectional mechanisms of macromolecular transport of the endoplasmic reticulum, mitochondria, peroxisomes and the nucleus, calls attention to the powerful experimental approaches which have been used for their elucidation, discusses their regulation and evolutionary origins, and highlights relatively unexplored areas.