RESUMEN
Efficient delivery of membrane proteins is a critical cellular process. The recently elucidated GET (Guided Entry of TA proteins) pathway is responsible for the targeted delivery of tail-anchored (TA) membrane proteins to the endoplasmic reticulum. The central player is the ATPase Get3, which in its free form exists as a dimer. Biochemical evidence suggests a role for a tetramer of Get3. Here, we present the first crystal structure of an archaeal Get3 homologue that exists as a tetramer and is capable of TA protein binding. The tetramer generates a hydrophobic chamber that we propose binds the TA protein. We use small-angle X-ray scattering to provide the first structural information of a fungal Get3/TA protein complex showing that the overall molecular envelope is consistent with the archaeal tetramer structure. Moreover, we show that this fungal tetramer complex is capable of TA insertion. This allows us to suggest a model where a tetramer of Get3 sequesters a TA protein during targeting to the membrane.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Archaea/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Archaea/química , Factores de Intercambio de Guanina Nucleótido/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The recently elucidated Get proteins are responsible for the targeted delivery of the majority of tail-anchored (TA) proteins to the endoplasmic reticulum. Get4 and Get5 have been identified in the early steps of the pathway mediating TA substrate delivery to the cytoplasmic targeting factor Get3. Here we report a crystal structure of Get4 and an N-terminal fragment of Get5 from Saccharomyces cerevisae. We show Get4 and Get5 (Get4/5) form an intimate complex that exists as a dimer (two copies of Get4/5) mediated by the C-terminus of Get5. We further demonstrate that Get3 specifically binds to a conserved surface on Get4 in a nucleotide dependent manner. This work provides further evidence for a model in which Get4/5 operates upstream of Get3 and mediates the specific delivery of a TA substrate.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Portadoras/química , Factores de Intercambio de Guanina Nucleótido/química , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Prueba de Complementación Genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana , Modelos Moleculares , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins.
Asunto(s)
Adenosina Trifosfatasas/química , Aspergillus fumigatus/enzimología , Factores de Intercambio de Guanina Nucleótido/química , Proteínas de la Fusión de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Fusión de la Membrana/química , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleótidos/química , Nucleótidos/metabolismo , Fenotipo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología Estructural de ProteínaRESUMEN
In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies.
Asunto(s)
Arginina/genética , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Pliegue de Proteína , Señales de Clasificación de ProteínaRESUMEN
Biosynthesis of membrane proteins requires that hydrophobic transmembrane (TM) regions be shielded from the cytoplasm while being directed to the correct membrane. Tail-anchored (TA) membrane proteins, characterized by a single C-terminal TM, pose an additional level of complexity because they must be post-translationally targeted. In eukaryotes, the GET pathway shuttles TA-proteins to the endoplasmic reticulum. The key proteins required in yeast (Sgt2 and Get1-5) have been under extensive structural and biochemical investigation during recent years. The central protein Get3 utilizes nucleotide linked conformational changes to facilitate substrate loading and targeting. Here we analyze this complex process from a structural perspective, as understood in yeast, and further postulate on similar pathways in other domains of life.