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Journal of Experimental Botany, Vol. 63, No. 4, pp. 1699–1712, 2012 doi:10.1093/jxb/err357 Advance Access publication 29 November, 2011 REVIEW PAPER Vipp1: a very important protein in plastids?! Ute C. Vothknecht1,*, Stephanie Otters1, Raoul Hennig2 and Dirk Schneider2,* 1 2 Department of Biology I, LMU Munich, D-82152 Planegg-Martinsried, Germany Department of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, D-55128 Mainz, Germany * To whom correspondence should be addressed. E-mail: [email protected] or [email protected] Received 5 September 2011; Revised 13 October 2011; Accepted 14 October 2011 Abstract As a key feature in oxygenic photosynthesis, thylakoid membranes play an essential role in the physiology of plants, algae, and cyanobacteria. Despite their importance in the process of oxygenic photosynthesis, their biogenesis has remained a mystery to the present day. A decade ago, vesicle-inducing protein in plastids 1 (Vipp1) was described to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Most follow-up studies clearly linked Vipp1 to membranes and Vipp1 interactions as well as the defects observed after Vipp1 depletion in chloroplasts and cyanobacteria indicate that Vipp1 directly binds to membranes, locally stabilizes bilayer structures, and thereby retains membrane integrity. Here current knowledge about the structure and function of Vipp1 is summarized with a special focus on its relationship to the bacterial phage shock protein A (PspA), as both proteins share a common origin and appear to have retained many similarities in structure and function. Key words: Chloroplasts, cyanobacteria, PspA, thylakoid membrane biogenesis, vesicle transport, Vipp1. Emergence of Vipp1 as a factor for thylakoid biogenesis Thylakoids are characteristic structures of cyanobacteria and chloroplasts, and the major complexes of oxygenic photosynthesis are embedded in these membranes. The thylakoid membrane system evolved in cyanobacteria in close correlation with the evolution of oxygenic photosynthesis and generated a novel bacterial compartment, the thylakoid lumen. Thylakoids are exclusively linked to organisms carrying out oxygenic photosynthesis, and the facility to build up and to modify such a complex membrane system has been transferred from the cyanobacterial ancestor to the plant cell during the endosymbiotic establishment of chloroplasts (reviewed in Vothknecht and Westhoff, 2001). Thylakoid membrane biogenesis is a multidimensional process, involving a concerted interplay of lipid, protein, and pigment synthesis, as well as their transport and integration into functional membrane-integral complexes. It is still unclear whether they assemble completely de novo or whether their membrane scaffold derives from the inner envelope of chloroplasts or the plasma membrane of cyanobacteria, respectively. While insertion of photosynthetic proteins into already existing thylakoid lipid bilayers has been analysed in some detail (reviewed in Aldridge et al., 2009), there is very little information about components involved in the formation of the thylakoid membrane itself (reviewed in Vothknecht and Westhoff, 2001; Nickelsen et al., 2010). The vesicle-inducing protein in plastids 1 (Vipp1) was first described in 1994 as a protein located in the chloroplasts of Pisum sativum (Li et al., 1994). Vipp1 is nuclear encoded and contains an N-terminal sequence sharing common characteristics with other chloroplast-directed transit peptides. Thus, like most plastidal proteins, Vipp1 is translated as a precursor on cytosolic ribosomes and is afterwards imported into the chloroplast by the TOC–TIC general import pathway (Schnell et al., 1997). An initial analysis further suggested that the protein is located at the inner envelope membrane as well as the thylakoids. Due to its localization and its mature size of 30 kDa, the protein was Abbreviations: PG, phosphatidyl glycerol; PS, photosystem; Psp, phage shock protein; Sec, general secretory pathway; Tat, twin-arginine translocation pathway; TIC, translocon on the inner chloroplast membrane; TOC, translocon on the outer chloroplast membrane; Vipp1, vesicle-inducing protein in plastids 1. ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 1700 | Vothknecht et al. first named M30 (membrane-associated protein of 30 kDa) (Li et al., 1994). However, Vipp1 does not display any features of intrinsic membrane proteins, as was revealed by structural analysis and supported by experimental approaches (Li et al., 1994; Kroll et al., 2001). Instead, further studies demonstrated a rather peripheral association of Vipp1 with chloroplast membranes. The unique localization of Vipp1 at the two membranes bordering the stromal compartment led to the presumption that Vipp1 might somehow be involved in the assembly of the thylakoid membrane system (Li et al., 1994). More precisely, it has been suggested that the protein is involved in the transport of lipids, which are synthesized at the chloroplast inner envelope. A potential involvement of Vipp1 in thylakoid membrane biogenesis was supported several years later by the drastic phenotype of an Arabidopsis thaliana high chlorophyll fluorescence mutant line (hcf155), in which the expression of the vipp1 gene was strongly reduced due to a T-DNA insertion in its promoter region (Kroll et al., 2001). Mutant plants expressed only about a fifth of the Vipp1 wild-type protein content under normal growth conditions and were incapable of autotrophic growth. Even on sugar-supplemented growth medium they showed a strong albinotic phenotype and did not survive beyond the stage of a small rosette (Kroll et al., 2001). Transmission electron microscopy of leaf sections revealed that mutant plants were not able to build a properly structured thylakoid membrane system. Their chloroplasts, while almost normal in size, contained very few internal membranes in rather severely distorted structures that are diffusely distributed inside the organelle. Correlating with their lack of thylakoid membranes, the capacity for oxygenic photosynthesis was extremely decreased (Kroll et al., 2001). Furthermore, hcf155 mutant plants displayed another phenotype in accordance with Vipp1’s alleged function in lipid transfer. Formation of vesicular structures at the inner envelope of chloroplasts could no longer be observed in the mutants, indicating that Vipp1 might be essential for this feature. Thus, the protein was renamed ‘vesicle-inducing protein in plastids 1’ (Kroll et al., 2001). At the same time, a vipp1 mutant of Synechocystis sp. PCC 6803 was described. While not fully segregated, mutant cells had a drastically reduced Vipp1 content. This cyanobacterial mutant displayed a similar phenotype to the hcf155 mutant line, showing a comparable loss of thylakoid membrane content and structure, as well as reduced photosynthetic activity (Westphal et al., 2001a). These findings not only supported the function of Vipp1 independently in two different organisms but also indicated a phylogenetically conserved function. PspA and Vipp1, a shared past (and present?) Phylogenetic analyses strongly support Vipp1 as a protein of prokaryotic origin that is found exclusively in cyanobacteria and chloroplasts (Li et al., 1994; Kroll et al., 2001; Westphal et al., 2001a; Fuhrmann et al., 2009a). This indicates a development in parallel to the emergence of the thylakoid membrane system and a connection to the evolution of oxygenic photosynthesis. However, proteins with significant similarities to Vipp1 have been identified in several non-photosynthetic bacteria (Fig. 1) in form of the phage shock protein A (PspA) (Brissette et al., 1990). PspA was first characterized following infection of Escherichia coli cells with filamentous phages, whereby the phage gene pIV protein was found to induce pspA expression to extremely high levels (Brissette et al., 1990). It was subsequently shown that PspA is a member of a bacterial stress response system, referred to as the ‘phage shock protein system’ (Psp system) (Brissette et al., 1991). The Psp system in E. coli responds not only to the invasion of filamentous phages but also to a number of different extracytoplasmic stress factors, such as heat, hyperosmotic conditions, organic solvents (Brissette et al., 1991; Weiner and Model, 1994; Kobayashi et al., 1998), inhibition of lipid biosynthesis (Bergler et al., 1994), and mislocalization or blockage of the protein export machinery (Kleerebezem and Tommassen, 1993; Kleerebezem et al., 1996; DeLisa et al., 2004). A common denominator of these stress conditions is the reduction of the proton motive force across the cytoplasmic membrane. In total, the Psp system in E. coli comprises seven psp genes. In addition to the pspABCDE operon, the transcriptional activator PspF as well as the effector protein PspG are encoded by separate genes (Brissette et al., 1991; Elderkin et al., 2002; Lloyd et al., 2004). Furthermore, the E. coli two-component system consisting of ArcA and ArcB has been shown to be involved in signal sensing and induction of the Psp-mediated stress response (Jovanovic et al., 2006, 2009). PspA is a hydrophilic protein of 25 kDa that, like Vipp1, shares no features of integral membrane proteins but appears to be peripherally associated with the cytoplasmic membrane of bacterial cells (Brissette et al., 1990; Cserzo et al., 1997; Kobayashi et al., 2007). As the most abundant protein and primary effector of the Psp response, PspA is responsible for several phenotypes connected to this system (Weiner and Model, 1994; Kleerebezem et al., 1996; DeLisa et al., 2004). It is assumed that PspA supports the bacterial cell in stress situations by blocking proton leakage (Kobayashi et al., 2007) or by inducing the down-regulation of proton motive force-consuming processes (Engl et al., 2009). The Psp system appears to be widespread throughout all bacterial domains. Psp proteins have been identified in Gram-negative and Gram-positive bacteria, as well as in archaea and cyanobacteria. In Yersinia enterocolitica, where the stress response is well established, the Psp system was found to be induced by two outer membrane secretins. Furthermore, it plays an important role in maintaining the membrane stability of the pathogen during cell infection (Darwin and Miller, 2001). Psp proteins are also highly expressed in Salmonella enterica (Eriksson et al., 2003) and Shigella flexneri (Lucchini et al., 2005) during macrophage infection. A functional analysis of the Psp response in the halophilite archaeon Halifax volcanii has pointed out that Vipp1: a very import protein in plastids | 1701 Fig. 1. Phylogenetic tree construction of PspA and Vipp1. Included in the analysis were proteins with sequenced similarity to PspA and Vipp1 from Actinomyces odontolyticus (Aco), Alteromonas macleodii (Alm), Anabaena variabilis (Anv1, Anv2), Arabidopsis thaliana (Ath), Arthrobacter chlorophenolicus (Arc), Azospirillum sp. (Azs), Bacillus sp. (Bas), Bacillus subtilis (Bacs), Bifidobacterium animalis (Bia), Bifidobacterium gallicum (Big), Burkholderia phymatum (Bup), Caldicellulosiruptor bescii (Cab), Caldicellulosiruptor owensensis (Cao), Candidatus Korarchaeum cryptofilum (Cac), Chlamydomonas reinhardtii (Chr), Chlorella variabilis (Chv), Chloroherpeton thalassium (Cht), Citrobacter sp. (Cis), Clostridium botulinum (Clb), Clostridium sp. (Cls), Crocosphaera watsonii (Crw1, Crw2), Cyanothece sp. (Cys1, Cys2), Delftia sp. (Des), Desulfatibacillum alkenivorans (Desa), Desulfobacterium autotrophicum (Dea), Desulvovibrio vulgaris RCH1 (Dev), Desulvovibrio vulgaris DP4 (Desv), Ectocarpus siliculosus (Ecs), Eikenella corrodens (Eic), Enterobacter sp. (Ens), Erythrobacter sp. (Ers), Escherichia coli (Esc), Gloeobacter violaceus (Glv), Gluconobacter oxydans (Glo), Glycine max (Glm), Haliangium ochraceum (Hao), Haloarcula marismortui (Ham), Haloquadratum walsbyi (Haw), Halorhabdus utahensis (Hau), Haloterrigena turkmenica (Hat), Heliobacterium modesticaldum (Hem), Hirschia baltica (Hib), Hordeum vulgare (Hov), Hypomonas neptunicum (Hyn), Klebsiella pneumoniae (Klp), Kytococcus sedentarius (Kys), Lyngbya majuscule (Lym1, Lym2), Lyngbya sp. (Lys1, Lys2), Micrococcus luteus (Mil), Microcoleus vaginatus (Miv1, Miv2) Micromonas pusilla (Mip), Micromonas spec. (Mis), Myxococcus xanthus (Myx), Natrialba magadii (Nam), Neisseria subflava (Nes), Nodularia spumigena (Nods1, Nods2), Nostoc punctiforme (Nop1, Nop2), Nostoc sp. (Nos1, Nos2) Novosphingobium sp. (Novs) Oryza sativa (Ors), Oscillatoria sp. (Oss1, Oss2), Phaeodactylum tricornutum (Pht), Photobacterium profundum (Phop), Phyllostachys edulis (Phe), Physcomitrella patens (Php), Picea sitchensis (Pics), Pisum sativum (Pis), Planctomyces brasiliensis (Plb), Planctomyces maris (Plm), Plesiocystis pacifica SIR-1 (Plp, Plep) Populus trichocarpa (Pot), Prochlorococcus marinus (Prm), Proteus mirabilis (Prs), Pseudomonas syringae (Pss), Renibacterium salmoninarum (Res), Rhodospirillum rubrum (Rhr), Ricinus communis (Ric), Ruminococcus sp. (Rus), Salmonella enterica (Sae), Selaginella moellendorffii (Smo), Shewanella baltica (Shb), Sorangium cellulosum (Soc), Sorghum bicolour (Sob), Sphingobium japonicum (Spj), Streptomyces lividans (Stl), Streptomyces pristinaespiralis (Stp), 1702 | Vothknecht et al. pspA expression was up-regulated in response to salt stress (Bidle et al., 2008). Phylogenetic analysis indicates that a minimal functional Psp system—containing just PspA, B, C, and F—is conserved in a-, d- and c-proteobacteria, whereas other Psp components show much less conservation or are even absent (Darwin, 2005). Moreover, PspA homologues also appear in organisms without connection to any other component of the Psp response, suggesting either a strong reduction of the Psp system or a newly acquired function of the remaining PspA homologue (reviewed in Darwin, 2005). In Streptomyces lividans, expression of pspa is strongly induced under membrane stress (Vrancken et al., 2008), which indicates that PspA has retained an important effector function in the stress response of this Gram-positive bacterium despite the loss of other Psp members. LiaH, the PspA homologue of the Gram-positive bacterium Bacillus subtilis (Mascher et al., 2004), belongs to the Lia system that is conserved in all Firmicutes, Bacillus, and Listeria species, where it regulates the bacterial stress response. The regulatory Lia system is comprised of the LiaRS two-component system linked to LiaF, a membrane-anchored negative regulator of LiaRdependent gene expression. The LiaRS two-component system recognizes different antibiotics and triggers a 100to 1000-fold induction of the liaIHGFSR operon. Beside a strong induction in response to external cell wall antibiotics that affect the lipid II cycle (Mascher et al., 2004; Pietiainen et al., 2005), expression of the Lia system is also induced by alkaline shock, as well as by treatment with organic solvents and secretion stress (Petersohn et al., 2001; Mascher et al., 2004; Hyyrylainen et al., 2005). The Lia system of B. subtilis thus seems to represent a complex regulatory stress response system in Gram-positive bacteria comparable with the Psp response system found in E. coli (Wolf et al., 2010). While most components of the Psp and Lia system differ, the primary target proteins of both systems, LiaH and PspA, show sequence and structural homology, indicating a similar mode of action. Many cyanobacterial genomes possess two genes encoding proteins with significant homology to members of the PspA/Vipp1 protein family (Fig. 1), even if they lack other genes encoding proteins of the original Psp response. Based on a phylogenetic analysis, these proteins divide into two different clades, suggesting that cyanobacteria require PspA as well as Vipp1. In the genome of Anabaena sp. PCC 7120, the genes encoding these two proteins are localized adjacent to each other with a gap of only 162 bp between the stop codon of the former and the start codon of the latter (Kaneko et al., 2001). A similar arrangement can be found in other cyanobacteria, suggesting that Vipp1 has evolved in an ancestor of today’s cyanobacteria from PspA by gene duplication and was passed to the plant lineage by the cyanobacterial endosymbiont (Westphal et al., 2001a). No pspA gene has been identified explicitly in any plant or alga, indicating that PspA was either absent in the original endosymbiont or was lost in the plant lineage shortly after the endosymbiotic event. While cyanobacterial PspA proteins cluster together with the Vipp1 branch containing both cyanobacterial and plant Vipp1 proteins, PspA proteins from bacteria other than cyanobacteria cluster in a separate clade, which also includes PspA proteins from Archaea (Fig. 1). This strongly supports the hypothesis that Vipp1 evolved from a cyanobacterial PspA rather than originating in a different bacterial ancestor. Initially, the situation appeared rather straightforward, with non-photosynthetic bacteria possessing only PspA, chloroplasts only Vipp1, and cyanobacteria encoding both proteins (Fig. 1). This observation linked the presence of Vipp1 to thylakoid membranes and supported a thylakoidspecific function. Such an assumption was further supported by the apparent lack of a vipp1 gene in the genome of Gloeobacter violoceus sp. PCC 7421 (Nakamura et al., 2003), a unicellular, obligate photoautotrophic bacterium that branches at an early stage of cyanobacterial evolution (Nelissen et al., 1995; Honda et al., 1999). It contains structural features that differ from those of common cyanobacteria, such as the lack of thylakoid membranes, and its photosynthetic machinery is located on the cytoplasmic membrane (Rippka et al., 1974). As Gloeobacter provides several primordial characteristics, the organism is an important tool to investigate the origin of oxygenic photosynthesis (Nakamura et al., 2003; Mimuro et al., 2008). In agreement with the alleged function of Vipp1 in thylakoid biogenesis, its absence in Gloeobacter provided a simple explanation for the inability of this organism to build up a functional thylakoid membrane system. Gloeobacter does contain a protein of the Vipp1/PspA family, which is annotated as a PspA homologue due to certain conserved structural characteristics (see below). However, in phylogenetic analysis, this Gloeobacter protein is placed on a separate side branch of the cyanobacterial Vipp1/PspA clade, so that a definite phylogenetic relationship to either PspA or Vipp1 cannot be obtained (Fig. 1). The availability of many more completely sequenced cyanobacterial genomes has caused the situation to become more complex. The genomes of several cyanobacteria, such as some Prochlorococcus species, also lack a gene encoding Vipp1, despite the presence of thylakoids in these organisms (Dufresne et al., 2003). In some of these cyanobacteria, the supposed lack of Vipp1 might be due to a misannotation of Synechococcus sp. (Syns1, Syns2), Synechocystis sp. (Sys1, Sys2), Thalassiosira pseudonana (Thp), Thiomonas intermedia (Thi), Trichodesmium erythraeum (Tre1a, Tre1b, Tre2), Vibrio sp. (Vibs), Vitis viniferum (Viv), Volvox carteri (Voc), Yersinia pestis (Yep), and Yersinia enterocolitica (Yee). Sequence alignments were obtained by Clustal 1.8 (Thompson et al., 1997). Phylogenetic tree construction was performed by maximum likelihood using Phyml 3.0 (Guindon and Gascuel, 2003). Transit sequences were omitted from the plant sequences for the analysis. Vipp1: a very import protein in plastids | 1703 the vipp1 genes as pspA, but in some of them Vipp1 indeed appears to be absent. While these findings question the direct correlation of Vipp1 to thylakoid membrane evolution, it is possible that these organisms have secondarily lost Vipp1 and now utilize a different system for thylakoid membrane biogenesis and/or maintenance. Since Vipp1 originated from PspA, the question arose as to what extent PspA function is still conserved in Vipp1 proteins. Expression of Vipp1 from the cyanobacterium Synechocystis sp. PCC6803 in an E. coli DpspA deletion strain prevented blockage of protein translocation (DeLisa et al., 2004), indicating that the cyanobacterial Vipp1 could somehow functionally replace the bacterial PspA. In contrast, endogenous PspA present in a Synechocystis Dvipp1 depletion strain was not sufficient to compensate the deficiency causing the drastic phenotype of those mutants, and thus to take over the function of Vipp1 (Westphal et al., 2001a; Fuhrmann et al., 2009b; Gao and Xu, 2009). Obviously, PspA is not necessary for the bacterial cells in the absence of stress factors, since E. coli DpspA mutants are perfectly viable (Brissette et al., 1991). In contrast, vipp1 mutants from Arabidopsis and Synechocystis show a dramatic phenotype even if vipp1 expression is not completely abolished but merely strongly decreased (Kroll et al., 2001; Westphal et al., 2001a; Aseeva et al., 2007; Gao and Xu, 2009). Along with the fact that many cyanobacteria still possess both proteins, there is strong evidence that Vipp1 proteins have gained a novel function in cyanobacteria and chloroplasts. However, Vipp1 has also been clearly correlated to stress response. In Synechocystis, Vipp1 was found amongst those plasma membrane proteins exhibiting the highest enhancements under high salt conditions (Huang et al., 2006). In contrast, the vipp1 gene was significantly down-regulated in the cold (Suzuki et al., 2001). Also, the Chlamydomonas Vipp1 was found in a group of proteins whose expression was up-regulated under high light (Im and Grossman, 2002). Nevertheless, in all these cases it is not clear whether the change in vipp1 expression represents a specific stress response or whether it is due to a general function of Vipp1 in thylakoid membrane synthesis and maintenance, which is affected under these conditions. PspA and Vipp1 proteins display certain conservation in primary amino acid sequence but, even in the same organism, such as in Synechocystis sp. PCC 6803, they share just ;30% sequence identity and 50% sequence similarity (Bultema et al., 2010). Nevertheless, all PspA/Vipp1 family members exhibit a common N-terminal core structure, the so-called PspA domain that is ;220 amino acids in length (Westphal et al., 2001a). Furthermore, the secondary and tertiary structures as well as the intrinsic propensity of PspA and Vipp1 to form higher ordered oligomers appear to be highly conserved (Aseeva et al., 2004; Hankamer et al., 2004; Fuhrmann et al., 2009a; Bultema et al., 2010). Based on computational predictions and spectroscopic measurements, an essentially exclusively a-helical structure was identified for the PspA-like domain of Vipp1/PspA proteins (Fig. 2A) (Hankamer et al., 2004; Fuhrmann et al., 2009a; Bultema et al., 2010). Only few amino acids within this region are predicted to be unstructured and to interrupt the helical domain. In contrast to PspA, Vipp1 proteins are longer due to a random coil spacer of variable size, connecting the PspA-like domain to a short a-helical domain of ;30 amino acids (Westphal et al., 2001a). This defined a-helical extension at the C-terminus appears to be a characteristic feature of Vipp1 proteins that clearly distinguishes Vipp1 from PspA. The a-helical regions of the PspA domain have a high propensity to form coiled-coil structures and to homooligomerize to higher ordered rings with molecular masses >1 MDa. While the exact tertiary structure of Vipp1 is presently unknown, large parts of the coiled-coil domain are The structure of Vipp1 Vipp1 proteins of plants and algae exhibit an N-terminal extension that has evolved as a transit peptide to ensure the import of the cytosolic-translated protein into the chloroplast (Li et al., 1994; Westphal et al., 2001a). Since the transit peptide is removed after translocation, it has no bearing on the structure of the mature protein and is therefore mostly excluded in structural comparisons between Vipp1 and PspA. While the phylogenetic relationship between Vipp1 and PspA is evident, it remains unclear to what extent the function of Vipp1 was altered in the course of evolution and which changes within the protein might affect any difference in function between the two proteins. Fig. 2. Vipp1 oligomer formation. (A) Schematic of the Synechocystis Vipp1 protein sequence and the positions of the helical domains (HDs) according to regions defined for E. coli PspA (Elderkin et al., 2005). Putative functions of the individual domains in oligomerization are indicated. Amino acid residues 222–267 represent the C-terminal extension typical for Vipp1 proteins. Predicted a-helical regions are indicated by grey bars. (B) Formation of Vipp1 rings from Vipp1 monomers involves intermediate dimeric, tetrameric, and string-like states. (C) Individual Vipp1 rings eventually interact to form double rings and rod-like structures. 1704 | Vothknecht et al. involved in oligomerization (Aseeva et al., 2004; Bultema et al., 2010). Based on structural analysis, PspA of E. coli was subdivided into four helical domains HD1–HD4 and, in a fragmentation approach, oligomerization of truncated E. coli PspA proteins was analysed in detail (Elderkin et al., 2005; Joly et al., 2009) (Fig. 2A). The centre regions HD2 and HD3 alone support formation of a PspA hexamer, whereas the HD1–HD2–HD3 protein forms dimers. Thus, while HD2 and HD3 are critical for hexamer formation, HD3 negatively influences hexamerization, thereby however stabilizing a dimeric PspA structure. HD4 supports formation of a PspA 36mer ring structure, and, thus, this domain appears to connect individual dimers. It might be assumed that similar regions control assembly and disassembly of Vipp1 oligomers in vivo, as indicated in Fig. 2A, but this has not been assessed in detail yet. Accessibility of the individual helical regions and their involvement in defined intermonomer contacts might be the key for controlling the actual oligomeric state of PspA/Vipp1 proteins. Chaperones might further be crucially involved in shielding defined regions of the protein for controlled Vipp1 assembly/disassembly (as discussed below). While the tertiary structures of PspA and Vipp1 are not well defined, the quaternary structures of both have been analysed to a certain degree in recent years. Both proteins form large homo-oligomeric rings, albeit with different dimensions (Aseeva et al., 2004; Hankamer et al., 2004; Liu et al., 2007; Fuhrmann et al., 2009a; Bultema et al., 2010). PspA from E. coli forms a single, distinct ring structure with a 9-fold rotational symmetry and a calculated mass of ;1 MDa. It has been suggested that this ring assembles from nine tetrameric building blocks (Hankamer et al., 2004). The PspA ring has an outer diameter of 20 nm, an inner diameter of 9.5 nm, and a height of ;8.5 nm (Hankamer et al., 2004). In contrast, Vipp1 proteins of Arabidopsis and Synechocystis organize into variable ring structures with molecular masses of up to ;2 MDa (Aseeva et al., 2004; Fuhrmann et al., 2009a). While for Synechocystis Vipp1 at least six different ring structures with a 12–17 internal rotational symmetry and outer diameters between 25 nm and 33 nm have been identified, all rings have a uniform height of 22 nm (Fuhrmann et al., 2009a). Based on recent results it seems likely that multiple copies of monomers (or smaller oligomeric building blocks) assemble into string-like structures representing intermediates during ring assembly or disassembly. Such an assumption is supported by the observation that some string-like structures have indeed been observed in the case of the Synechocystis protein. Furthermore, controlled disassembly of Vipp1 results in formation of distinct tetrameric and dimeric (dis)assembly intermediates, indicating that Vipp1 dimers are the minimal building blocks of the Vipp1 rings (Fuhrmann et al., 2009a). At higher concentrations, individual Vipp1 rings eventually stack together and form barrel-like double-ring structures, as observed with Vipp1 from cyanobacteria and higher plants (Aseeva et al., 2004; Fuhrmann et al., 2009a). Such double-ring structures potentially represent an assembly intermediate during formation of multiple-ring stacking structures with a rodlike shape, as detected after heterologous expression and in vitro refolding of the Chlamydomonas reinhardtii Vipp1 (Liu et al., 2007). Such scaffold-like structures have also been described for PspA (Standar et al., 2008). Figure 2B and C summarize all presently identified Vipp1 structures and a potential assembly/disassembly pathway. The characteristic C-terminal extension of Vipp1 proteins does not appear to be essential for the formation of rings or rods (Aseeva et al., 2004; Liu et al., 2007). Fusing a protein to the Vipp1 C-terminus does not interfere either with its oligomerization propensity or with thylakoid biogenesis in Arabidopsis (Aseeva et al., 2007). Thus, the C-terminus is probably not crucially involved in formation of Vipp1 oligomers and a free C-terminus is not essential for Vipp1 activity. However, as the C-terminal extension appears to be a characteristic feature of Vipp1, it is crucial for discriminating PspA from the PspA-like Vipp1 proteins. Based on a sequence alignment of Vipp1 homologues of cyanobacteria, algae, and higher plants, the following consensus sequences of the Vipp1 C-terminus was generated: V/I/L-D/E-X-E-L-E/D-E/D-L-R/(K)-R/K-X-L/I-D/ N-X-(L)-X3 (Fig. 3A). Even though the amino acids are not strictly conserved at most positions, the properties of individual amino acid side chains are preserved at various places. The Vipp1 C-terminus is predicted to form an amphiphilic a-helix with one non-polar, hydrophobic face, containing two acidic residues at the N-terminus and two basic residues at the C-terminus (Fig. 3B). As the C-terminus most probably protrudes out of the ring structures (Aseeva et al., 2004), these conserved features of the Vipp1 C-terminal helix eventually allow and control defined interactions with proteins, lipids, or membranes. Structural dynamics, interactions, and functions Most data somehow link both PspA and Vipp1 to membranes. In chloroplasts, Vipp1 is associated with the inner envelope as well as with thylakoid membranes, and in cyanobacteria the protein localizes in close vicinity to both the cytoplasmic and thylakoid membranes (Srivastava et al., 2005; Fuhrmann et al., 2009a). Detailed studies on membrane attachment revealed that the PspA protein of E. coli is able to bind directly to membrane lipids, having a clear preference for lipids with negatively charged head groups (Kobayashi et al., 2007). In vivo, however, PspA is mainly recruited to membranes by the membrane-integral proteins PspB and C, and it has been suggested that these proteins sense stress signals and modulate PspF activity by a controlled interaction with PspA (Joly et al., 2010). Only upon release of PspA from the PspBC complex the interaction of PspA with the transcription activator PspF permitted. This releases the suppression of the psp operon by PspF and results in overexpression of PspA. Subsequently, PspA binds to membranes directly for its function in stress protection. Vipp1: a very import protein in plastids | 1705 Fig. 3. Consensus sequence of the Vipp1 C-terminus. (A) Alignment of Vipp1 sequences from cyanobacteria, green algea, brown algae, and plants revealed a conserved consensus sequence within the last 44 amino acids, forming the a-helical Vipp1 C-terminus. The degree of conservation is shown by the number of bits (Crooks et al., 2004). The approach disclosed a strict conservation of side chain properties in several positions, even if the amino acids were not conserved. (B) Helical wheel projection of the consensus sequence underlined in A predicts the formation of an amphiphatic a-helix with a hydrophilic face on one side of the helix (indicated). Furthermore, the helix is bookended by two conserved acidic residues at the N-terminus and two basic residues at the C-terminus. Amino acid properties are indicated: magenta, acidic residue; blue, basic residue; black/grey, non-polar residue. Different oligomeric structures of PspA appear to have different functions in E. coli. When interacting with PspF, PspA is present as a hexamer, whereas formation of the PspA 36mer appears to be required for direct membrane attachment and maintenance of membrane integrity. In contrast to PspA, no hexameric Vipp1 structure has been reported, and, as Vipp1 does not seem to interact with any component similar to PspF, a hexameric assembly stage might not exist. Extraction of Vipp1 from cyanobacterial thylakoids furthermore suggests that the protein binds as rings to membranes (Fuhrmann et al., 2009a). Soluble Vipp1 rings have been identified in the cyanobacterial cytoplasm, which might be less stable than membranebound rings (Fuhrmann et al., 2009a). While for PspA membrane attachment appears to be regulated by the oligomeric state, it is not clear how this is regulated for Vipp1. It is noteworthy that in cyanobacteria and plants, two Vipp1 forms showing an ;2 kDa difference in apparent molecular mass have been identified (Kroll et al., 2001; Aseeva et al., 2004; Liu et al., 2005; Fuhrmann et al., 2009a). While the difference between these two Vipp1 forms has not been determined yet, Vipp1 might be post-translationally modified. Thus, the two Vipp1 forms observed on SDS–gels might represent two structurally different Vipp1 populations having different membrane binding affinities. Individual Vipp1 monomers have an intrinsic propensity to homo-oligomerize and to aggregate. In an Arabidopsis mutant strain containing a massively decreased Vipp1 content, oligomers have not been observed, and thus it seems that a critical Vipp1 concentration is needed for complex formation (Aseeva et al., 2007). At higher concentrations, rod-like structures form, which have been suggested to represent a chloroplastic microtubule-like system (Liu et al., 2007). Rod-like structures of unknown origin have indeed been observed in chloroplasts as well as in cyanobacteria (Anderson et al., 1973; Lawrence et al., 1984; van de Meene et al., 2006), and it is therefore intriguing to speculate that such cytoskeleton-like elements could be used as tracks for a directed transport of proteins and/or vesicles to distinct locations within chloroplasts or cyanobacteria. Nevertheless, while Vipp1 rings stack at higher concentrations, no rod structures have been reported thus far for PspA proteins. Formation of rods, which are essentially identical to Vipp1 rods, is also observed in vitro by the ringforming, membrane-integral Holine protein of bacteriophage k (Savva et al., 2008). Since artificial formation of 1706 | Vothknecht et al. the identified rod structures here strongly depended on the experimental conditions, the existence of Vipp1 rods in vivo still has to be proven unambiguously. Oligomerization and membrane attachment of Vipp1 might be controlled by a chaperone-mediated disassembly of Vipp1 rings. Shielding defined regions of the PspA domain would allow controlled assembly/disassembly of Vipp1 rings (compare Fig. 2A) and consequently membrane binding of the protein. Vipp1 of the green alga C. reinhardtii interacts with the chloroplast chaperone Hsp70B, as well as with the Hsp40 homologue CDJ2 (Liu et al., 2005). Binding of these chaperones to Vipp1 resulted in disassembly of large Vipp1 rings and rods in vitro (Liu et al., 2007), although it is not yet clear whether the rings disassemble into monomers or to lower ordered oligomeric states. Thus, the Hsp70/Hsp40 system might be part of a cellular system which controls assembly/disassembly of Vipp1 oligomers and thereby its membrane attachment. Binding to chloroplast chaperones might also be an early step in Vipp1 import into the organelle, to prevent unspecific aggregation of the protein and to control oligomer formation (Fig. 4). Such an assumption is supported by the recent observation that Vipp1 can be co-precipitated together with components of the TIC– TOC protein import machinery of pea chloroplasts (Jouhet and Gray, 2009). While a single Hsp70 and Hsp40 protein has been identified in the stroma of Chlamydomonas chloroplasts (Liu et al., 2005), multiple Hsp70/DnaK and Hsp40/DnaJ proteins are encoded in cyanobacteria (Nimura et al., 2001; Rupprecht et al., 2007, 2010; Sato et al., 2007; Duppre et al., 2011). It is thus not clear yet which DnaK/DnaJ pair might regulate assembly/disassembly of Vipp1 rings in cyanobacteria. However, involvement of a special cyanobacterial Fig. 4. Import and assembly of Vipp1 in chloroplasts. Pre-Vipp1 is imported from the cytoplasm via the translocons at the outer and inner envelope of chloroplasts (TOC and TIC) into the chloroplast stroma. Molecular chaperones prevent misfolding and unspecific aggregation. Oligomerization of Vipp1 is guided by the Hsp70 and Hsp40 chaperones in an ATP-dependent manner. Oligomeric Vipp1 rings eventually interact with the inner envelope (IE) or thylakoid membranes (TM). DnaK and DnaJ protein in thylakoid membrane biogenesis has been suggested (Nimura et al., 1996; Oguchi et al., 1997), and thus it is intriguing to speculate that these proteins interact with Vipp1. Such an assumption is supported by the observation that the specified DnaK and DnaJ chaperones are not encoded in the genome of the thylakoid-free cyanobacterium Gloeobacter (Nakamura et al., 2003). Hsp90C has also been reported to form a complex with Hsp70, Hsp40, and Vipp1 in Clamydomonas chloroplasts (Heide et al., 2009). It is noteworthy that an Arabidopsis hsp90C mutant line has defects in chloroplast development and thylakoid stacking (Cao et al., 2003). The point mutation in Hsp90C in this mutant might affect interaction of chloroplast Hsp90 with Vipp1 (Heide et al., 2009), which subsequently perturbs thylakoid membrane biogenesis. Involvement of Vipp1 in the integration of thylakoid membrane proteins has been suggested based on the observation of a direct interaction of Vipp1 with the Albino3.2 protein in Chlamydomonas chloroplasts (Gohre et al., 2006). The thylakoid-localized Albino3.2 belongs to the conserved YidC/Oxa1p/Alb3 protein family, whose members function as insertases, chaperones, and/or assembly factors for transmembrane proteins (reviewed in Wang and Dalbey, 2010). It has been suggested that Vipp1 is either involved in membrane integration of Albino3.2 or that Vipp1 delivers substrates to Albino3.2 for membrane integration (Gohre et al., 2006). Albino3.2 has an essential role during assembly of photosystem II (PSII) and PSI reaction centres in thylakoid membranes (Gohre et al., 2006). During Albino3.2-mediated membrane integration and/or assembly of protein complexes, the membrane structure might be locally perturbed and Vipp1 could eventually be required to stabilize the membrane. In Chlamydomonas, depletion of the Albino3.2 protein results in overproduction of Vipp1 (Gohre et al., 2006), while depletion of YidC in E. coli causes an increased expression of PspA (van der Laan et al., 2003). Thus, Vipp1 might be involved in stabilizing the membrane structure during Albino3.2-mediated protein insertion but also participates in maintaining the membrane integrity after Albino3.2 depletion. Vipp1 (additionally?) could associate with the TIC complex to stabilize the inner envelope membrane in the vicinity of the TIC complex during TICmediated protein integration. Involvement of PspA-like proteins in protein translocation/integration is further supported by the observation that increased PspA expression has been observed in E. coli cells having a defect in the Sec pathway (Kleerebezem and Tommassen, 1993). Furthermore, overexpression of PspA in S. lividans improved the function of both the Sec and Tat pathway (Vrancken et al., 2007). PspA has been suggested to minimize proton leakage of the bacterial plasma membrane during Tat-mediated protein translocation/integration, and thus the occurrence of Vipp1 and/or PspA has been linked to the Tat pathway of bacteria, archaea, and plant chloroplasts (DeLisa et al., 2004). Interestingly, this function can be taken over by the full-length Vipp1 as well as by a C-terminally truncated Vipp1 of Synechocystis PCC 6803 in an E. coli pspA deletion strain (DeLisa et al., 2004). Vipp1: a very import protein in plastids | 1707 Thylakoid membranes, the photosynthetic electron transfer chain, and Vipp1 An increased amount of thylakoid membranes and chlorophylls correlates with increased light harvesting, and chloroplasts adapt to high-light growth conditions by reduction of thylakoid membranes (Anderson et al., 1973; Lichtenthaler et al., 1981; Melis and Harvey, 1981). Furthermore, in both chloroplasts and cyanobacteria, the ratio of the two photosystems, PSI and PSII, alters in response to changing light conditions (Chow et al., 1990; Kim et al., 1993; Murakami et al., 1997). A comparative proteomic analysis of bundle sheet and mesophyll chloroplasts in maize has shown that mesophyll cell plastids, which contain a larger thylakoid membrane system, have a 10-fold increased Vipp1 content (Majeran et al., 2005). Mesophyll cells also contain high amounts of PSII, which is mostly lacking in bundle sheet chloroplasts. Depletion of Vipp1 in both higher plants and cyanobacteria resulted in a massive decrease of assembled thylakoid membrane layers and in defects in the photosynthetic electron transfer chain (Kroll et al., 2001; Westphal et al., 2001a). In Arabidopsis the defect could not be attributed to a deficiency of a single photosynthetic complex, but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain (Kroll et al., 2001). Vipp1-depleted plants displayed a dramatic reduction of both photosystems as well as of the cytochrome b6f complex. A disturbed energy transfer between antenna and photosynthetic core complexes has also been noticed, and the light-harvesting complexes are not properly attached to the photosystems (Aseeva et al., 2007). Electron microscopic analysis of Synechocystis Dvipp1 mutant cells showed a similar reduction in thylakoid membranes. Furthermore, the overall thylakoid membrane morphology was disturbed and the thylakoid membranes were less well arranged than in the wild-type strain (Fuhrmann et al., 2009b). This observation indicates that the composition of the thylakoid membranes and/or thylakoid membrane modelling is affected by Vipp1 depletion. In cyanobacteria, the two photosystems typically form dimers and trimers, respectively, and trimerization of PSI was affected by Vipp1 depletion (Fuhrmann et al., 2009b). Furthermore, the relative PSI:PSII ratio was also significantly decreased in the depletion strain, which indicates an impaired activity of the photosynthetic electron transfer chain (Schneider et al., 2001, 2004; Volkmer et al., 2007). After controlled depletion of Vipp1 in Synechocystis a drastic effect on photosynthetic complexes has been observed (Gao and Xu, 2009), which further suggests a direct effect of Vipp1 on photosystem biogenesis, assembly, or function. Vipp1 and the need for vesicles in chloroplasts and cyanobacteria The debate on how thylakoid membranes are formed in mature chloroplasts without a connection to the inner envelope has been longstanding (reviewed in Vothknecht and Westhoff, 2001). Several of the thylakoid constituents, such as monogalactosidyl diacylglycerol and other lipids, are synthesized within the inner envelope (Douce, 1974; Joyard and Stumpf, 1981; Kobayashi et al., 2007). Consequently, there is a need for regulated transport between internal membranes within chloroplasts. This might be achieved by a vesicle transport system of eukaryotic origin that was suggested to exist in chloroplasts of plants (reviewed in von Wettstein, 2001). Vesicle formation can be observed in chloroplasts of higher plants under certain experimental conditions (Westphal et al., 2001b) but is no longer detected in the Arabidopsis Vipp1 depletion strain (Kroll et al., 2001). It is indeed conceivable that Vipp1 of higher plants might support thylakoid biogenesis and maintenance by sustaining vesicle traffic and thereby ensuring the flow of lipids and other non-lipid components from the inner envelope to the thylakoids. It has long been debated whether thylakoid membranes also represent a completely independent internal membrane system in cyanobacteria or whether they are still linked to the cytoplasmic membrane. However, recent structural analyses strongly suggest that thylakoids indeed are not directly connected to cyanobacterial cytoplasmic membranes, and thus represent a separate internal membrane entity (Liberton et al., 2006; van de Meene et al., 2006; Nevo et al., 2007; Schneider et al., 2007). Furthermore, it has been suggested that precursors of the cyanobacterial photosystems are pre-assembled within the plasma membrane (Zak et al., 2001; Keren et al., 2005), implying the existence of a distinct lipid and protein transfer system from the cytoplasmic to the thylakoid membrane. Such transfer might involve vesicles or a temporary connection of the membrane systems. Thylakoid centres in cyanobacteria may be the origin of thylakoids, as these regions appear to connect thylakoid membranes with the cytoplasmic membrane in some cyanobacteria (Nickelsen et al., 2010). However, thylakoid centre structures are not typical and highly conserved in these organisms, indicating that they are not generally involved in thylakoid membrane biogenesis. Vesicle accumulation was detected in chloroplasts of A. thaliana and P. sativum by transmission electron microscopy while providing conditions that are known to prevent vesicle fusion in the cytosol. These include a decrease in the temperature or treatment with toxins such as ophiobolin A and microcystin, established inhibitors of cytosolic proteins involved in this process (Morre et al., 1991; Kroll et al., 2001; Westphal et al., 2001b). Until now, no vesicle transport system has been observed under those conditions in cyanobacteria, green algae, or even charophytes, implying that the specific vesicle transport system of chloroplasts might be restricted to embryophytes (bryophytes, pteridophytes, and spermatophytes) and has evolved somewhere in an ancestor between today’s charyophytes and bryophytes (Westphal et al., 2003). Nevertheless, this has been a point of ongoing debate, with different experimental evidence supporting the occurrence of vesicle transport in photosynthetic organisms other than embryophytes (Hoober 1708 | Vothknecht et al. et al., 1991). After staining the cyanobacterial cytoplasmic membrane with a fluorescent dye, intracellular vesicular structures appeared within individual cells (Schneider et al., 2007). Also, in the cyanobacterium Microcoleus sp., large vesicular structures have been identified by electron microscopy (Nevo et al., 2007). Vipp1 might have a function similar to coat proteins in the secretory pathway, which stabilize highly bent membranes during vesicle budding. For E. coli PspA it has been observed that it predominantly binds to the cell poles of the bacterium, where the membranes are highly bent and where membrane stress and proton leakage might be higher than in planar bilayer regions (Engl et al., 2009). Thus, Vipp1 might be involved in stabilizing highly bent membranes, as is the case during vesicle formation. The diverse oligomeric Vipp1 ring structures could also be involved in vesicle constriction, similar to dynamins in eukaryotes (Fuhrmann et al., 2009a). While assembly and disassembly of dynamin is regulated by its GTPase domain, controlled disassembly and formation of smaller Vipp1 rings eventually involves Hsp70 and Hsp40, as discussed above. PspA and Vipp1 show homology to the eukaryotic Snf7 protein, which is involved in protein sorting and transport from eukaryotic endosomes to the vacuole/lysosome, and all three proteins are clustered in a single protein family in the pfam database (Finn et al., 2009). Furthermore, proteins with homologies to components of the vesicular transfer system of the secretory pathway have been predicted to localize to chloroplasts, and some of the predicted proteins also have clear homologues in cyanobacteria (Andersson and Sandelius, 2004). Thus, it cannot be excluded per se that a vesicular transfer system exists in chloroplasts and cyanobacteria. Vipp1 function: does a common theme emerge? All studies carried out in chloroplasts and cyanobacteria indicate that Vipp1 binds directly to membranes, locally stabilizes bilayer structures, and thereby retains membrane integrity. While initial observations indicated an involvement of Vipp1 in vesicle formation in chloroplasts, the observed perturbations in an Arabidopsis vipp1 depletion strain might be caused by an altered membrane structure and/or composition. However, also during vesicle formation, membranes deform and Vipp1 might have a function in stabilizing bent membranes, similar to coat proteins. Involvement of Vipp1 in vesicle transfer between internal membrane systems in chloroplasts and cyanobacteria is still an appealing hypothesis as a general function of Vipp1, since a membrane chaperone role could most probably also be fulfilled by PspA, which is still present in cyanobacteria. Nevertheless, Vipp1 has some distinct features different from PspA. While both PspA and Vipp1 form ring structures, the ring dimensions are dramatically different and this leads to the necessity for variance in the PspA and Vipp1 tertiary structure. PspA monomers most probably form a kinked helix bundle, whereas Vipp1 may have a long stretched a-helical shape (Bultema et al., 2010). These different quaternary structures of PspA and Vipp1 eventually determine the exact physiological functions of the individual proteins. A key to the specific function of Vipp1 could also be the C-terminal extension unique to Vipp1 proteins. This extension appears to correlate with the appearance of thylakoid membranes, as Gloeobacter contains neither Vipp1 nor thylakoid membranes, whereas it encodes a PspA protein, which could be crucially involved in maintaining the integrity of the cytoplasmic membrane. It is noteworthy that in the genome of Gloeobacter, genes involved in sulphoquinovosyl diacylglycerol synthesis are also missing (Nakamura et al., 2003). Sulphoquinovosyl diacylglycerol is a constituent of typical thylakoid membranes, and, like phosphatidyl glycerol (PG), contains a negatively charged head group. Depletion of Vipp1 in Synechocystis results in a decreased PSI content and in a reduced trimerization propensity of PSI (Fuhrmann et al., 2009b), a phenotype also observed after PG depletion (Hagio et al., 2000; Sato et al., 2000). Also in Arabidopsis PG is required for photosynthesis, and a mutant having inactivated PG synthesis was no longer able to grow photoautotrophically and showed a severe reduction in thylakoid membrane (Hagio et al., 2002). As PspA from E. coli has been shown to interact preferentially with the head groups of negatively charged lipids (Kobayashi et al., 2007), all these observations link PspA and Vipp1 not only generally to membranes, but more specifically to negatively charged membrane lipids. 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