RESUMEN
PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestructura , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Liposomas Unilamelares/metabolismoRESUMEN
Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Familia de Multigenes , Nostoc/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Pollos , Microscopía por Crioelectrón , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Evolución Molecular , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestructura , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas de la Membrana , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Tilacoides/metabolismo , Proteínas Bacterianas/genética , Cianobacterias/metabolismo , Fusión de Membrana/fisiología , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/genética , Membranas/metabolismo , Unión Proteica/fisiología , Protones , Synechocystis/metabolismoRESUMEN
The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+ However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of â¼1 mm Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Magnesio/metabolismo , Fusión de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Plastidios/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismo , Magnesio/química , Plastidios/química , Unión Proteica , Synechocystis/crecimiento & desarrollo , Tilacoides/químicaRESUMEN
IM30/Vipp1 proteins are crucial for thylakoid membrane biogenesis in chloroplasts and cyanobacteria. A characteristic C-terminal extension distinguishes these proteins from the homologous bacterial PspA proteins, and this extension has been discussed to be key for the IM30/Vipp1 activity. Here we report that the extension of the Synechocystis IM30 protein is indispensable, and argue that both, the N-terminal PspA-domain as well as the C-terminal extension are needed in order for the IM30 protein to conduct its in vivo function. In vitro, we show that the PspA-domain of IM30 is vital for stability/folding and oligomer formation of IM30 as well as for IM30-triggered membrane fusion. In contrast, the IM30 C-terminal domain is involved in and necessary to stabilize defined contacts to negatively charged membrane surfaces, and to modulate the IM30-induced membrane fusion activity. Although the two IM30 protein domains have distinct functional roles, only together they enable IM30 to work properly.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Membranas/metabolismo , Tilacoides/metabolismo , Cloroplastos/metabolismo , Unión Proteica/fisiología , Dominios Proteicos , Synechocystis/metabolismoRESUMEN
The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Cloroplastos/metabolismo , Cinética , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfatidilgliceroles/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Resonancia por Plasmón de Superficie , Synechocystis/genética , Synechocystis/metabolismo , Tilacoides/metabolismoRESUMEN
The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30kDa, a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg2+ is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas Bacterianas/química , Proteínas de la Membrana/química , Fosfolípidos/química , Synechocystis/química , Tilacoides/química , Arabidopsis/metabolismo , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Cationes Bivalentes , Citoesqueleto/química , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Magnesio/química , Magnesio/metabolismo , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Biogénesis de Organelos , Fosfolípidos/metabolismo , Fotosíntesis/fisiología , Células Vegetales/química , Células Vegetales/metabolismo , Células Vegetales/ultraestructura , Multimerización de Proteína , Synechocystis/metabolismo , Synechocystis/ultraestructura , Tilacoides/metabolismo , Tilacoides/ultraestructuraRESUMEN
Cyanobacteria carry out oxygenic photosynthesis and share many features with chloroplasts, including thylakoid membranes, which are mainly composed of membrane lipids and protein complexes that mediate photosynthetic electron transport. Although the functions of the various thylakoid protein complexes have been well characterized, the details underlying the biogenesis of thylakoid membranes remain unclear. Galactolipids are the major constituents of the thylakoid membrane system, and all the genes involved in galactolipid biosynthesis were recently identified. In this chapter, I summarize recent advances in our understanding of the factors involved in thylakoid development, including regulatory proteins and enzymes that mediate lipid biosynthesis.
Asunto(s)
Cianobacterias/metabolismo , Galactolípidos/biosíntesis , Tilacoides/metabolismo , Secuencia de Carbohidratos , Datos de Secuencia Molecular , Oxígeno/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Especificidad por Sustrato , Tilacoides/químicaRESUMEN
Thylakoids of land plants have a bipartite structure, consisting of cylindrical grana stacks, made of membranous discs piled one on top of the other, and stroma lamellae which are helically wound around the cylinders. Protein complexes predominantly located in the stroma lamellae and grana end membranes are either bulky [photosystem I (PSI) and the chloroplast ATP synthase (cpATPase)] or are involved in cyclic electron flow [the NAD(P)H dehydrogenase (NDH) and PGRL1-PGR5 heterodimers], whereas photosystem II (PSII) and its light-harvesting complex (LHCII) are found in the appressed membranes of the granum. Stacking of grana is thought to be due to adhesion between Lhcb proteins (LHCII or CP26) located in opposed thylakoid membranes. The grana margins contain oligomers of CURT1 proteins, which appear to control the size and number of grana discs in a dosage- and phosphorylation-dependent manner. Depending on light conditions, thylakoid membranes undergo dynamic structural changes that involve alterations in granum diameter and height, vertical unstacking of grana, and swelling of the thylakoid lumen. This plasticity is realized predominantly by reorganization of the supramolecular structure of protein complexes within grana stacks and by changes in multiprotein complex composition between appressed and non-appressed membrane domains. Reversible phosphorylation of LHC proteins (LHCPs) and PSII components appears to initiate most of the underlying regulatory mechanisms. An update on the roles of lipids, proteins, and protein complexes, as well as possible trafficking mechanisms, during thylakoid biogenesis and the de-etiolation process complements this review.
Asunto(s)
Embryophyta/fisiología , Embryophyta/ultraestructura , Tilacoides/fisiología , Tilacoides/ultraestructura , Embryophyta/crecimiento & desarrollo , Organogénesis de las PlantasRESUMEN
Intracellular compartmentalization is a hallmark of eukaryotic cells. Dynamic membrane remodeling, involving membrane fission/fusion events, clearly is crucial for cell viability and function, as well as membrane stabilization and/or repair, e.g., during or after injury. In recent decades, several proteins involved in membrane stabilization and/or dynamic membrane remodeling have been identified and described in eukaryotes. Yet, while typically not having a cellular organization as complex as eukaryotes, also bacteria can contain extra internal membrane systems besides the cytoplasmic membranes (CMs). Thus, also in bacteria mechanisms must have evolved to stabilize membranes and/or trigger dynamic membrane remodeling processes. In fact, in recent years proteins, which were initially defined being eukaryotic inventions, have been recognized also in bacteria, and likely these proteins shape membranes also in these organisms. One example of a complex prokaryotic inner membrane system is the thylakoid membrane (TM) of cyanobacteria, which contains the complexes of the photosynthesis light reaction. Cyanobacteria are evolutionary closely related to chloroplasts, and extensive remodeling of the internal membrane systems has been observed in chloroplasts and cyanobacteria during membrane biogenesis and/or at changing light conditions. We here discuss common principles guiding eukaryotic and prokaryotic membrane dynamics and the proteins involved, with a special focus on the dynamics of the cyanobacterial TMs and CMs.
Asunto(s)
Cianobacterias , Células Eucariotas , Eucariontes , Cianobacterias/metabolismo , Cloroplastos/metabolismo , TilacoidesRESUMEN
IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósido-Trifosfatasa/metabolismo , Synechocystis/enzimología , Tilacoides/enzimología , Adenosina Trifosfato/química , Proteínas Bacterianas/genética , Clonación Molecular , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólisis , Cinética , Proteínas de la Membrana/genética , Microscopía Electrónica , Nucleósido-Trifosfatasa/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Synechocystis/genética , Synechocystis/ultraestructura , Tilacoides/genética , Tilacoides/ultraestructuraRESUMEN
The inner membrane-associated protein of 30 kDa (IM30, also known as Vipp1) is required for thylakoid membrane biogenesis and maintenance in cyanobacteria and chloroplasts. The protein forms large rings of â¼2 MDa and triggers membrane fusion in presence of Mg2+. Based on the here presented observations, IM30 rings are built from dimers of dimers, and formation of these tetrameric building blocks is driven by interactions of the central coiled-coil, formed by helices 2 and 3, and stabilized via additional interactions mainly involving helix 1. Furthermore, helix 1 as well as C-terminal regions of IM30 together negatively regulate ring-ring contacts. We propose that IM30 rings represent the inactive form of IM30, and upon binding to negatively charged membrane surfaces, the here identified fusogenic core of IM30 rings eventually interacts with the lipid bilayer, resulting in membrane destabilization and membrane fusion. Unmasking of the IM30 fusogenic core likely is controlled by Mg2+, which triggers rearrangement of the IM30 ring structure.
RESUMEN
The inner membrane-associated protein of 30 kDa (IM30), also known as the vesicle-inducing protein in plastids 1 (Vipp1), is essential for photo-autotrophic growth of cyanobacteria, algae and higher plants. While its exact function still remains largely elusive, it is commonly accepted that IM30 is crucially involved in thylakoid membrane biogenesis, stabilization and/or maintenance. A characteristic feature of IM30 is its intrinsic propensity to form large homo-oligomeric protein complexes. 15 years ago, it has been reported that these supercomplexes have a ring-shaped structure. However, the in vivo significance of these ring structures is not finally resolved yet and the formation of more complex assemblies has been reported. We here present and discuss research on IM30 conducted within the past 25 years with a special emphasis on the question of why we potentially need IM30 supercomplexes in vivo.
RESUMEN
Biogenesis and dynamics of thylakoid membranes likely involves membrane fusion events. Membrane attachment of the inner membrane-associated protein of 30 kDa (IM30) affects the structure of the lipid bilayer, finally resulting in membrane fusion. Yet, how IM30 triggers membrane fusion is largely unclear. IM30 monomers pre-assemble into stable tetrameric building blocks, which further align to form oligomeric ring structures, and differently sized IM30 rings bind to membranes. Based on a 3D reconstruction of IM30 rings, we locate the IM30 loop 2 region at the bottom of the ring and show intact membrane binding but missing fusogenic activity of loop 2 mutants. However, helix 7, which has recently been shown to mediate membrane binding, was located at the oppossite, top side of IM30 rings. We propose that a two-sided IM30 ring complex connects two opposing membranes, finally resulting in membrane fusion. Thus, IM30-mediated membrane fusion requires a Janus-faced IM30 ring.
Asunto(s)
Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/metabolismo , Tilacoides/ultraestructura , Liposomas/metabolismo , Fusión de Membrana , Modelos Moleculares , Unión Proteica , Multimerización de ProteínaRESUMEN
Photosynthetic membranes, or thylakoids, are the most extensive membrane system found in the biosphere. They form flattened membrane cisternae in the cytosol of cyanobacteria and in the stroma of chloroplasts. The efficiency of light energy capture and conversion, critical for primary production in ecosystems, relies on the rapid expansion of thylakoids and their versatile reorganization in response to light changes. Thylakoid biogenesis results from the assembly of a lipid matrix combined with the incorporation of protein components. Four lipid classes are conserved from cyanobacteria to chloroplasts: mono- and digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidyldiacylglycerol. This review focuses on the production and biophysical properties of galactolipids, making them determinant factors for the nonvesicular/nonlamellar biogenesis and for the three-dimensional architecture of nascent thylakoids. The regulation of MGD1, the committing enzyme of galactolipid biosynthesis in Arabidopsis, via feedback regulatory loops and control of protein binding to membranes, is also detailed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotosíntesis/fisiología , Células Vegetales/metabolismo , Tilacoides/metabolismoRESUMEN
Chlorophylls (Chl) in photosynthetic apparatuses, along with other macromolecules in chloroplasts, are known to undergo degradation during leaf senescence. Several enzymes involved in Chl degradation, by which detoxification of Chl is safely implemented, have been identified. Chl degradation also occurs during embryogenesis and seedling development. Some genes encoding Chl degradation enzymes such as Chl b reductase (CBR) function during these developmental stages. Arabidopsis mutants lacking CBR (NYC1 and NOL) have been reported to exhibit reduced seed storability, compromised germination, and cotyledon development. In this study, we examined aberrant cotyledon development and found that NYC1 is solely responsible for this phenotype. We inferred that oxidative damage of chloroplast membranes caused the aberrant cotyledon. To test the inference, we attempted to trans-complement nyc1 mutant with overexpressing VIPP1 protein that is unrelated to Chl degradation but which supports chloroplast membrane integrity. VIPP1 expression actually complemented the aberrant cotyledon of nyc1, whereas stay-green phenotype during leaf senescence remained. The swollen chloroplasts observed in unfixed cotyledons of nyc1, which are characteristics of chloroplasts receiving envelope membrane damage, were recovered by overexpressing VIPP1. These results suggest that chloroplast membranes are a target for oxidative damage caused by the impairment in Chl degradation. Trans-complementation of nyc1 with VIPP1 also suggests that VIPP1 is useful for protecting chloroplasts against oxidative stress.
RESUMEN
The Vesicle Inducing Protein in Plastids 1 (Vipp1) was suggested to be involved in thylakoid membrane formation in both chloroplasts and cyanobacteria. The protein shows sequence homology to the Phage Shock Protein A (PspA) from bacteria, and both proteins have similar secondary structures. 2D-structures of PspA and of Vipp1 have been determined by electron microscopy in the recent years. Both PspA and Vipp1 form large homooligomeric rings with high molecular masses but their ring dimensions differ significantly. Furthermore, Vipp1 forms rings with different rotational symmetries whereas PspA appears to form rings with singular rotational symmetry. In this article addendum we compare the structures of PspA and Vipp1. Furthermore, we suggest a spatial structural model of the observed Vipp1 rings.