Lipoprotein biogenesis in Gram- positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em Matthew I. Hutchings 1 , Tracy Palmer 2 , Dean J. Harrington 3 and Iain C. Sutcliffe 4 1 School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK 2 Division of Molecular and Environmental Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK 3 Division of Biomedical Sciences, School of Life Sciences, University of Bradford, West Yorkshire, BD7 1DP, UK 4 Biomolecular and Biomedical Research Centre, School of Applied Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST, UK Gram-positive bacterial lipoproteins are a functionally diverse and important class of peripheral membrane proteins. Recent advances in molecular biology and the availability of whole genome sequence data have overturned many long-held assumptions about the export and processing of these proteins, most notably the recent discovery that not all lipoproteins are exported as unfolded substrates through the general secretion pathway. Here, we review recent discoveries concerning the export and processing of these proteins, their role in virulence in Gram-positive bacteria and their potential as vaccine candidates or targets for new anti- microbials. Bacterial lipoproteins Lipoproteins in Gram-positive bacteria are cell envelope proteins anchored into the outer leaflet of the plasma membrane. Lipid modification is achieved through covalent addition of a diacylglyceride to an indispensable cysteine residue in the lipoprotein signal peptide, as origin- ally described for the prototypical Braun’s lipoprotein of Escherichia coli [1]. This provides a common anchoring mechanism for what is now recognized to be an abundant and functionally diverse class of peripheral membrane proteins. In Gram-positive bacteria, lipoproteins function within a subcellular region that is defined at its inner aspect by the plasma membrane and at its outer aspect by the peptidoglycan and other layers of the cell wall. Lipoproteins of Gram-positive bacteria have, thus, been proposed to be functional equivalents of periplasmic proteins in Gram-negative bacteria, a comparison that is most directly sustained by the fact that, in Gram-positive bacteria, substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters are typically lipoproteins [2,3]. Moreover, cell fractionation experiments and recent advances in electron microscopy have lent some credibility to the controversial concept of a Gram-positive ‘periplasm’ [4–6]. Cryoelectron microscopy has also provided evidence supporting the presence of an outer membrane per- meability barrier in the mycolic acid-containing actinomy- cete bacteria Mycobacterium smegmatis, Mycobacterium bovis and Corynebacterium glutamicum [7,8]. In addition, since the last review of Gram-positive lipoproteins 13 years ago [3] our understanding of the diverse functions of these proteins has been greatly advanced by the availability of whole genome sequence data. These advances, along with important new insights into the lipoprotein biogenesis pathway, make it timely to revisit this subject. Crossing the cytoplasmic membrane Almost all exported proteins are transported across the cytoplasmic membrane of prokaryotes by one of two dis- tinct export pathways. The general secretory (Sec) path- way is the predominant route of protein transport [9]. The Sec machinery recognizes proteins bearing N-terminal signal peptides (Figure 1a) and transports them across the membrane in an unfolded conformation. By contrast, the more recently discovered Tat (twin arginine protein transport) system transports folded and even oligomeric proteins, which often bind redox cofactors [10]. Proteins are also targeted to the Tat system by means of N-terminal signal peptides, which in this case harbour an almost invariant and essential twin-arginine motif (Figure 1b). Exported proteins that are destined to become lipidated contain a motif in their signal peptides known as a lipobox, which directs them to the lipoprotein biogenesis machinery after transport (Figure 1). It had long been assumed, based primarily on studies in E. coli, that all lipoprotein precur- sors are synthesized with signal peptides that direct them to the Sec pathway for translocation across the cytoplasmic membrane in an unfolded state [1,11]. More recently, it has also become clear that some putative lipoproteins can be translocated utilizing the SecA2-dependent accessory Sec pathway, which is found in some, but not all, Gram- positive bacteria [12,13]. The first indication that some lipoprotein precursors could be exported in a fully folded state through Tat came during an analysis of the dimethyl- sulphoxide (Dms) reductase in the Gram-negative bacter- ium Shewenella oneidensis. DmsA was shown to contain a Tat signal sequence with a lipobox and to be translocated via Tat in a complex with its partner subunit DmsB [14]. The DmsB subunit lacks a signal sequence and the proteins must, therefore, fold and form a complex before export. DmsA is a relatively rare example of an outer Review Corresponding author: Hutchings, M.I. (m.hutchings@uea.ac.uk). 0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.10.001 Available online 6 December 2008 13