RESUMO
Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported via other pathways. While targeting signals for each route have common overall characteristics, individual signal peptides vary greatly in their amino acid sequences. How do these diverse signals interact specifically with the proteins that comprise the appropriate transport machinery and, at the same time, avoid targeting to an alternate route? The recent publication of the crystal structures of components of the Sec transport machinery now allows a more thorough consideration of the interactions of signal sequences with these components.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Partícula de Reconhecimento de Sinal/genética , Transdução de Sinais/genéticaRESUMO
Like prokaryotic Sec-dependent protein transport, chloroplasts utilize SecA. However, we observe distinctive requirements for the stimulation of chloroplast SecA ATPase activity; it is optimally stimulated in the presence of galactolipid and only a small fraction of anionic lipid and by Sec-dependent thylakoid signal peptides but not Escherichia coli signal peptides.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Cloroplastos , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Galactolipídeos/química , Lipídeos/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Fosfatidilgliceróis/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sinais Direcionadores de Proteínas/genética , Canais de Translocação SEC , Proteínas SecARESUMO
SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the signal peptide binding groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53 residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acid residues 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Marcadores de Fotoafinidade/metabolismo , Sinais Direcionadores de Proteínas , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biotina/química , Biotina/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Canais de Translocação SEC , Proteínas SecARESUMO
Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas Motores Moleculares/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Dimerização , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC , Proteínas SecARESUMO
SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third alpha-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of 2.4 microM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Sinais Direcionadores de Proteínas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Temperatura Alta , Sondas Moleculares , Canais de Translocação SEC , Proteínas SecA , Espectrometria de FluorescênciaRESUMO
Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fenilalanina/análogos & derivados , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biotina/metabolismo , Reagentes de Ligações Cruzadas , Proteínas de Escherichia coli/química , Hidrólise , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fenilalanina/metabolismo , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Raios UltravioletaRESUMO
In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.