Protein Secretion Pathways in Bacteria PDF
Preview Protein Secretion Pathways in Bacteria
Protein Secretion Pathways in Bacteria Protein Secretion Pathways in Bacteria Edited by Bauke Oudega Vrije Universiteit Amsterdam, Department of Molecular Microbiology, The Netherlands SPRINGER-SCIENCE+BUSINESS MEDIA, B.V A c.I.P. Catalogue record for this book is available from the Library of Congress. ISBN 978-94-010-3974-1 ISBN 978-94-010-0095-6 (eBook) DOI 10.1007/978-94-010-0095-6 Printed an acid-free paper Ali Rights Reserved © 2003 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 2003 Softcover reprint ofthe hardcover lst edition 2003 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. TABLE OF CONTENTS Preface Vll 1. Protein Targeting to the Inner Membrane Joen Luirink and Bauke Oudega 1 2. The SEC Translocase Chris van den Does, Nico Nouwen and Arnold J.M. Driessen 23 3. The TAT Protein Export Pathway Tracy Palmer and Ben C. Berks 51 4. Assembly of Inner Membrane Proteins in Escherichia coli David Drew, Linda Froderberg, Louise Baars, Joen Luirink and Jan-Willem de Gier 65 5. Biogenesis of Outer Membrane Proteins Jan Tommassen and Rome Voulhoux 83 6. Chaperones and Folding Catalysts Involved in the General Protein Secretion Pathway of Escherichia coli Nellie Harms and Hans de Cock 99 7. Type I Protein Secretion Systems in Gram-Negative Bacteria: Escherichia coli a-Hemolysin Secretion Ivaylo Gentschev and Werner Goebel 121 8. Type II Protein Secretion Alain Filloux and Manon Gerard-Vincent 141 9. The Type III Secretion Pathway in Pathogenic Bacteria Claude Parsot 167 10. Autotransporters BenR. Otto 191 v 11. Assembly of Adhesive Organelles on Gram-Negative Bacteria Sheryl S. Justice, Karen W. Dodson, Matthew R. Chapman, Michelle M. Barnhart and Scott J. Hultgren 207 12. Export of Bacteriocins Bauke Oudega 233 13. Biogenesis of Flagella: Export of Flagellar Proteins via the Flagellar Machine Tohru Minamino and Shin-Ichi Aizawa 249 14. Protein Secretion in Gram-Positive Bacteria Rob Meima and Jan Maarten van Dijl 271 vi PREFACE The passed decades have seen an explosive growth in our understanding of the properties and functions ofliving cells. Especially our knowledge of the various components and complex structures of bacterial cells increased enormously. The genomes of over 50 different bacterial species have been sequenced. We have not yet identified all the different gene products of, for instance, Escherichia coli or Bacillus subtilis, but it will not be to long before we know most of the gene products, their cellular location, structure and function, and their specific role in one or more of the cellular processes. Some people even begin to dream about a complete picture of the functioning of a simple, living, unicellular organism, to understand life itself! This book plays a part in that dream, that beautiful future perspective of Biology. The book provides an broad overview of how bacterial cells are organized, how proteins are targeted out of the bacterial cytosol and end up in the inner membrane, the periplasm or even in the outer membrane. In addition, several chapters deal with mechanisms by which proteins are released into the extracellular environment. Many inspiring scientists have contributed to the book. Each of them is well known is his or her field of research. I like to thank them all for their invaluable contribution and their continuing research efforts in my area of research, the bacterial cell surface. In my work as a teacher and researcher so far, I have been looking for a book like this one, a book dealing with all different aspects of protein targeting, membrane insertion, folding and secretion. I hope that this first edition can inspire numerous students and young scientist to use the knowledge presented, to continue investigating all the marvelous aspects of the bacterial cell and the intriguing mechanisms and molecular machines that are used by bacterial cells. I also hope that teachers can use the book in their courses, especially in the area of molecular microbiology, medical microbiology and biotechnology. Bauke Oudega vii Chapter 1 PROTEIN TARGETING TO THE INNER MEMBRANE Joen Luirink and Bauke Oudega Department of Molecular Microbiology, IMBWlBioCentrum Amsterdam Faculty of Biology, Vrije Universiteit Amsterdam De Boelelaan 1087 1081 HV Amsterdam, NL 1. INTRODUCTION Bacteria appear to be simply organized organisms, but bacterial cells do have different subcellular compartments: the cytoplasm, the cytoplasmic or inner membrane (IM), the cell wall and in the case of gram-negative bacteria, the periplasm and the outer membrane (OM). Proteins are synthesized in the cytosol, and a large number of these proteins fold in the cytoplasm and play their role in this compartment. However, another large number of proteins (about 40 % of the total amount of proteins) is destined for functioning in one of the extra-cytoplasmic environments. These proteins have to be targeted to the 1M, during or after completion of translation, and they have to be inserted into or translocated across this membrane. The mechanisms by which extra-cytoplasmic proteins are inserted into or translocated across the 1M, transported to their final destination, fold and function, are described in more detail in one of the following chapters of this book. This chapter will deal with the first steps in these translocation pathways: the early recognition of membrane proteins and of secreted proteins by various targeting factors, and their targeting to generic or specific protein translocation sites (translocons) in the 1M of Escherichia coli. B. Oudega (ed.), Protein Secretion Pathways in Bacteria, 1-21. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 2 2. TRANSLOCONSAS RECEPTORS FOR TARGETING COMPLEXES The E. coli 1M contains different types of translocons (Fig. 1). The best studied and most widely used is the so-called Sec-translocon which appears to function both in secretion and in membrane integration of proteins. It consists of several integral membrane proteins that are thought to form a proteinaceous channel, and a peripheral bound ATPase to drive the translocation reaction (reviewed in Fekkes and Driessen, 1999). The core of this multisubunit complex is formed by SecY, SecE and SecG together with the peripherally bound SecA. This core complex is also referred to as translocase to underscore its catalytic activity. In addition, under certain experimental conditions three accessory proteins can be co-purified with the core translocon. These proteins are SeeD, SecF and YajC. They apparently increase the efficiency of the translocation reaction although their exact role is not clear (Fekkes and Driessen, 1999). "spontaneous" post- co folded proteins translational translational yid C moderately strongly twin-Arg 5S hydrophobic S5 hydrophobic ss Fig. 1. Schematic overview of the different protein translocation pathways present in the E. coli inner membrane. Some characteristic features are indicated. SecA is an important component of the Sec translocase. It binds to almost all components that are involved in the translocation process, exhibits ATPase activity, and regulates its expression by binding to its own mRNA (reviewed in Driessen et at., 1995). Most importantly for this 3 chapter, it also functions in the reception of secretory proteins that are being targeted to the translocase (see below as well as chapter 2). When bound to the membrane at the SecYEG complex, SecA is "activated" for the high affmity recognition of the SecB export chaperone (Hartl et ai., 1990), and of targeting signals in secreted proteins. The Sec-translocon is the common target of two distinct targeting pathways that pilot 1M and secreted proteins to the membrane. 1M proteins are in general targeted co-translationally to the Sec-translocon by the so called SRP (Signal Recognition Particle) targeting route (detailed below). The signal that is recognized in this route is a hydrophobic targeting peptide that is most often found at the N-terminus of the protein. In contrast, most periplasmic and OM proteins as well as some proteins that are secreted into the extracellular environment are targeted post-translationally (or late co translationally) to the Sec-translocon via the so-called SecB targeting route (detailed below). These latter proteins are synthesized with an N-terminal cleavable signal peptide or signal sequence. Recently, a new accessory translocon component has been identified, YidC, that appears to operate exclusively in the integration of membrane proteins perhaps as an intermediate beween the aqueous Sec translocon and the hydrophobic lipid bilayer (Scotti, 2000; see also chapter 4). Interestingly, YidC also acts independent of the Sec translocon either as a separate entity or in a different, yet unknown translocon. This "independent" YidC translocon is thought to facilitate membrane insertion of a subset ofIM proteins (Samuelson et ai., 2000; FrOderberg et ai., 2001). In addition to the Sec-machinery, another protein translocation pathway has been discovered. This pathway is used by proteins that are characterized by a so-called twin-arginine motif in their N -terminal targeting signal (TAT pathway, reviewed in Berks et ai., 2000). The TAT pathway appears to function specifically in the translocation of folded proteins like enzymes that contain a cofactor. So far, specific cytoplasmic targeting factors that play a targeting role in this route have not been identified. Details of this pathway will be described in chapter 3. Besides the Sec and TAT translocons, other specific targeting and secretion routes exist that are used by proteins that have to be translocated across the entire cell envelope. Examples are the so-called type I, type III and IV routes, the flagellum biosynthesis route as well as the bacteriocin release route. These routes will be described in more detail in one of the following chapters of this book. Finally, it can not be excluded that some 1M proteins integrate into the membrane spontaneously, i.e. without the aid of a protein machinery. 4 3. TARGETING SIGNALS The targeting signals present in 1M proteins as well as in secreted proteins, that mediate targeting by the main SRP and SecB targeting pathways to the Sec-translocon will be described in this section. Signals for targeting of most secreted proteins are located both in a cleavable, hydrophobic N-terminal extension of the pre-secretory protein (preprotein) as well as in the mature region. The N-terminal extention is called signal sequence, signal peptide or leader peptide. The signal sequence is cleaved (processed) during passage of the 1M by a signal peptidase (see chapter 2). 1M proteins are sometimes targeted by a cleavable signal sequence just like secreted proteins. More often, however, targeting is achieved by a signal sequence that is not cleaved and also serves to anchor the protein in the 1M. This type of targeting signal is called "signal anchor sequence" (SA) to emphasize its dual function. 3.1 Signal sequence The N-terminal signal sequence of prep rote ins ranges in length from about 20 to 30 amino acid residues. It is characterized by a positively charged N-terminal region (about 6 residues), a hydrophobic central part or core region (10-15 residues), and a polar C-terminal region (about 6 residues) that precedes the cleavage site of the signal peptidase (von Heijne, 1985) (Fig. 2). Signal sequences show no primary sequence similarity but have similar physical characteristics and are often functionally interchangeable between different organisms (von Heijne, 1990). These shared features are reflected by common mechanisms for recognition and functioning of signal sequences both in targeting and membrane insertion. c N H + mature domain -3 -1 Fig. 2. Schematic presentation of a signal sequence of a preprotein. N, amino-terminal, positively charged region; H, hydrophobic, central region; C, carboxyl-terminal region containing rather well conserved amino acids with short neutral side chains at position -1 and -3. The arrow indicates the cleavage site.
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