RESUMEN
BACKGROUND: Most tail-anchored (TA) membrane proteins are delivered to the endoplasmic reticulum through a conserved posttranslational pathway. Although core mechanisms underlying the targeting and insertion of TA proteins are well established in eukaryotes, their role in mediating TA protein biogenesis in plants remains unclear. We reported the crystal structures of algal arsenite transporter 1 (ArsA1), which possesses an approximately 80-kDa monomeric architecture and carries chloroplast-localized TA proteins. However, the mechanistic basis of ArsA2, a Get3 (guided entry of TA proteins 3) homolog in plants, for TA recognition remains unknown. RESULTS: Here, for the first time, we present the crystal structures of the diatom Pt-Get3a that forms a distinct ellipsoid-shaped tetramer in the open (nucleotide-bound) state through crystal packing. Pulldown assay results revealed that only tetrameric Pt-Get3a can bind to TA proteins. The lack of the conserved zinc-coordination CXXC motif in Pt-Get3a potentially leads to the spontaneous formation of a distinct parallelogram-shaped dimeric conformation in solution, suggesting a new dimer state for subsequent tetramerization upon TA targeting. Pt-Get3a nonspecifically binds to different subsets of TA substrates due to the lower hydrophobicity of its α-helical subdomain, which is implicated in TA recognition. CONCLUSIONS: Our study provides new insights into the mechanisms underlying TA protein shielding by tetrameric Get3 during targeting to the diatom's cell membrane.
Asunto(s)
Diatomeas , Diatomeas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Multimerización de ProteínaRESUMEN
Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.
Asunto(s)
Dominio AAA , Adenosina Trifosfatasas , Adenosina Trifosfatasas/química , Secuencia Conservada , Hidrólisis , Proteínas de la Membrana/química , Multimerización de ProteínaRESUMEN
Homologs of the protein Get3 have been identified in all domains yet remain to be fully characterized. In the eukaryotic cytoplasm, Get3 delivers tail-anchored (TA) integral membrane proteins, defined by a single transmembrane helix at their C terminus, to the endoplasmic reticulum. While most eukaryotes have a single Get3 gene, plants are notable for having multiple Get3 paralogs. Get3d is conserved across land plants and photosynthetic bacteria and includes a distinctive C-terminal α-crystallin domain. After tracing the evolutionary origin of Get3d, we solve the Arabidopsis thaliana Get3d crystal structure, identify its localization to the chloroplast, and provide evidence for a role in TA protein binding. The structure is identical to that of a cyanobacterial Get3 homolog, which is further refined here. Distinct features of Get3d include an incomplete active site, a "closed" conformation in the apo-state, and a hydrophobic chamber. Both homologs have ATPase activity and are capable of binding TA proteins, supporting a potential role in TA protein targeting. Get3d is first found with the development of photosynthesis and conserved across 1.2 billion years into the chloroplasts of higher plants across the evolution of photosynthesis suggesting a role in the homeostasis of photosynthetic machinery.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fotosíntesis , Adenosina Trifosfatasas/metabolismo , Embryophyta , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode "anion transporter ATPase" homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined.IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Animales , Proteínas de Transporte de Anión/genética , Femenino , Genoma Bacteriano , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Operón , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Get3 in yeast or TRC40 in mammals is an ATPase that, in eukaryotes, is a central element of the GET or TRC pathway involved in the targeting of tail-anchored proteins. Get3 has also been shown to possess chaperone holdase activity. A bioinformatic assessment was performed across all domains of life on functionally important regions of Get3 including the TRC40-insert and the hydrophobic groove essential for tail-anchored protein binding. We find that such a hydrophobic groove is much more common in bacterial Get3 homologs than previously appreciated based on a directed comparison of bacterial ArsA and yeast Get3. Furthermore, our analysis shows that the region containing the TRC40-insert varies in length and methionine content to an unexpected extent within eukaryotes and also between different phylogenetic groups. In fact, since the TRC40-insert is present in all domains of life, we suggest that its presence does not automatically predict a tail-anchored protein targeting function. This opens up a new perspective on the function of organellar Get3 homologs in plants which feature the TRC40-insert but have not been demonstrated to function in tail-anchored protein targeting. Our analysis also highlights a large diversity of the ways Get3 homologs dimerize. Thus, based on the structural features of Get3 homologs, these proteins may have an unexplored functional diversity in all domains of life.
Asunto(s)
Adenosina Trifosfatasas/química , ATPasas Transportadoras de Arsenitos/química , Evolución Molecular , Factores de Intercambio de Guanina Nucleótido/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , ATPasas Transportadoras de Arsenitos/genética , ATPasas Transportadoras de Arsenitos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Bombas Iónicas/química , Bombas Iónicas/genética , Bombas Iónicas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In mammals and yeast, tail-anchored (TA) membrane proteins destined for the post-translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well-known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane-trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide-free open state and bound to adenylyl-imidodiphosphate. This approximately 80-kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA-binding groove comprise the interlocking hook-like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.
Asunto(s)
Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Retículo Endoplásmico/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways.
Asunto(s)
Adenosina Trifosfatasas/química , Evolución Molecular , GTP Fosfohidrolasas/química , Factores de Intercambio de Guanina Nucleótido/química , Nucleotidasas/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Archaea/clasificación , Archaea/genética , Archaea/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , GTP Fosfohidrolasas/clasificación , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Nucleotidasas/clasificación , Nucleotidasas/genética , Nucleotidasas/metabolismo , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Proteins destined for the endomembrane system of eukaryotic cells are typically translocated into or across the membrane of the endoplasmic reticulum and this process is normally closely coupled to protein synthesis. However, it is becoming increasingly apparent that a significant proportion of proteins are targeted to and inserted into the ER membrane post-translationally, that is after their synthesis is complete. These proteins must be efficiently captured and delivered to the target membrane, and indeed a failure to do so may even disrupt proteostasis resulting in cellular dysfunction and disease. In this review, we discuss the mechanisms by which various protein precursors can be targeted to the ER and either inserted into or translocated across the membrane post-translationally. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.