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Proteins to be secreted from eukaryotic cells are delivered to the extracellular space after trafficking through a secretory pathway composed of several complex intracellular compartments. Secretory proteins are first translocated from the cytosol into the endoplasmic reticulum (ER), after which they travel by vesicular trafficking via various intermediate destinations en route to the plasma membrane where they are released from the cell by exocytosis. By sharp contrast, secretion in prokaryotes involves the translocation of proteins directly across the plasma membrane. While these two systems are superficially dissimilar, they are evolutionarily and mechanistically related. This relationship between the prokaryotic and eukaryotic systems of secretion forms the backdrop for this chapter focused on protein translocation into the ER. In the first part of this chapter, the essential steps and core machinery of ER translocation are discussed relative to evolutionarily conserved principles of protein secretion. The last section then explores the concept of regulation, a poorly understood facet of translocation that is argued to be evolutionarily divergent, relatively specific to the ER, and likely to be most highly developed in metazoans.

The Sec translocase or translocon is the essential and ubiquitous system for protein translocation across or into the membrane. The core channel, the SecYE complex, is conserved across biological kingdoms and most of the polypeptide chains which are routed to extracellular or membrane locations in Bacteria use this pathway. Biochemical and genetic approaches have yielded a substantial body of information about functional aspects of Sec-mediated translocation and this information has recently been enriched with structural data at atomic resolution. This chapter reviews previously acquired facts and concepts concerning the Sec translocase of Bacteria in light of recent structural results and considers implications of these findings.

While the process of protein translocation has been extensively addressed in Bacteria and Eukarya, little is known of how proteins cross the membranes of Archaea, the third domain of Life. Analysis thus far suggests the hybrid-like nature of the archaeal protein translocation system, combining selected aspects of the bacterial and eukaryal processes together with Archaea-specific features. The archaeal translocation apparatus simultaneously incorporates homologues of system components found either in Bacteria or Eukarya but not in both, yet seemingly does not include other important elements of these two systems. Moreover, certain facets of the archaeal protein translocation process appear specific to this domain, possibly reflecting adaptations to the extreme environments in which Archaea exist.

Protein transport through and into biological membranes is a process of fundamental importance in all living organisms. In eukaryotes, protein translocation through the endoplasmic reticulum is carried out by a membrane protein complex called Sec61, usually while associated with ribosomes. In Bacteria and Archaea, protein translocation through and into the cytosolic membrane is conducted by the homologous SecY complex. In , SecYEG consists of three polypeptides associating with either ribosomes or the partner ATPase SecA, which drive the translocation reaction. The structure of the SecY complex has been determined in a closed conformation. SecY encapsulates the central protein channel formed by the two halves of the subunit, closed by a short plug domain and a ring of hydrophobic residues. In combination with previous results, the atomic structure has led to models of how the complex might move during the reaction cycle, but events and conformational changes associated with the engagement of substrate and the partner protein are not understood. This chapter will review the recent structural results relating to the protein-conducting channel. The implications of these findings will be described in the context of the mechanism through which proteins pass across and into the membrane. The nature of the interaction with substrate and translocation partners will be discussed together with the possible movements that occur during the reaction cycle.

Membrane proteins are inserted into the lipid bilayer in Bacteria by two pathways. The Sec machinery is responsible for the insertion of the majority of the membrane proteins after targeting by the SRP/FtsY components. However, there is also a class of membrane proteins that insert independent of the Sec machinery. These proteins require a novel protein called YidC. Recently, the structural details of the Sec machinery have come to light via X-ray crystallography. There are now structures of the membrane-embedded Secprotein-conducting channel, the SecA ATPase motor, and the targeting components. Structural information gives clues to how a polypeptide is translocated across the membrane and how the transmembrane segments of a membrane protein are released from the Sec complex. Additionally, the structures of the targeting components shed light on how substrates are selected for transport and delivered to the membrane.

The twin-arginine transport (Tat) system is a protein-targeting pathway found in the cytoplasmic membranes of many eubacteria, some Archaea, and the chloroplasts and mitochondria of plants. It is apparendy not a feature of animal physiology. Substrate proteins are targeted to a membrane-bound transport apparatus by N-terminal signal peptides harbouring a distinctive ‘twin-arginine’ amino acid sequence motif, and, most remarkably, all substrate proteins are transported in a fully folded conformation. Model systems most commonly used to study the fundamentals of Tat transport are the Gram-negative eubacterium , the Gram-positive eubacterium , and thylakoid membranes derived from pea or maize chloroplasts. Here, we have attempted to integrate our knowledge of the key aspects of these well-characterized Tat protein transport pathways, to carve-out some shared principles between systems, and arrive at a broad consensus covering the physiology and biochemistry of Tat transport.

Many proteins synthesised in the cytosol are translocated across or inserted into the endoplasmic reticulum (ER) membrane. These proteins include not only those resident in the ER itself, but others destined for post-ER destinations such as the Golgi complex, lysosomes or secretion into the extracellular environment. Proteins that fail to fold or assemble correctly are detected by the quality control system of the ER and are disposed of by a process known as ER-associated degradation. Degradation does not occur in the ER itself. Rather the aberrant proteins are exported from the ER for degradation by the ubiquitin/proteasome pathway in the cytosol. This involves the retro-translocation of these proteins across the ER membrane. In this chapter we discuss our current understanding of the process of retro-translocation.

Plastids, exemplified by chloroplasts, are a diverse group of essential organelles that distinguish plant cells. The biogenesis of these organelles is essential to plant growth and development, and relies on the import of >2500 nuclear-encoded proteins from the cytoplasm. The import of the large majority of these proteins is dependent on the Toe-Tic machinery of the chloroplast envelope. However, an ever increasing number of new pathways for targeting proteins to numerous chloroplast sub-compartments have been identified. Furthermore, it appears that the multiple targeting pathways and the regulation of import play direct roles in the differentiation and specific functions of distinct plastid types during plant growth and development. This chapter summarizes the state of the field, emphasizing the mechanisms of targeting proteins to and across the plastid envelope, and to chloroplast sub-compartments.

Mitochondria are surrounded by a double-membrane system that defines four intra-organelle compartments: the outer membrane, the inner membrane, the intermembrane space and the matrix. Hundreds of nuclear-encoded mitochondrial proteins are synthesized as precursor proteins in the cytosol and have to be targeted to and imported into the mitochondria. To facilitate this import process, precursor proteins contain targeting and sorting sequences which are recognized and decoded by mitochondrial translocation machineries. This chapter describes the mechanisms by which mitochondrial precursor proteins are targeted to the mitochondria, and sorted into the correct sub-mitochondrial compartment.

Peroxisomes are organelles equipped with enzymes for lipid metabolism and hydrogen-peroxide-based respiration. Though many details of their metabolism are understood today, basic aspects concerning their biogenesis, including translocation of peroxisomal proteins into and through the peroxisomal membrane, still remain unknown. Nevertheless, the past years have brought forth a wealth of detailed information on the proteins required for proper biogenesis of peroxisomes. This review focuses on the basic principles and on recent developments in the field of peroxisome biogenesis. More comprehensive or specialized reviews can be found in the reference list.