RESUMEN
Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine. However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway.
Asunto(s)
Proteínas de Arabidopsis , Proteínas Bacterianas , Núcleo Celular/genética , Cianobacterias/citología , Proteínas de Escherichia coli , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Pisum sativum/citología , Plastidios/genética , Tilacoides/genética , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Arabidopsis/genética , Arginina/genética , Arginina/metabolismo , Evolución Biológica , Transporte Biológico , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Cianobacterias/genética , Eucariontes/citología , Eucariontes/genética , Concentración de Iones de Hidrógeno , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Pisum sativum/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/enzimología , Plastidios/metabolismo , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Canales de Translocación SEC , Proteína SecA , Tilacoides/enzimología , Tilacoides/metabolismoRESUMEN
A subset of lumen proteins is transported across the thylakoid membrane by a Sec-independent translocase that recognizes a twin-arginine motif in the targeting signal. A related system operates in bacteria, apparently for the export of redox cofactor-containing proteins. In this report we describe a key feature of this system, the ability to transport folded proteins. The thylakoidal system is able to transport dihydrofolate reductase (DHFR) when an appropriate signal is attached, and the transport efficiency is almost undiminished by the binding of folate analogs such as methotrexate that cause the protein to fold very tightly. The system is moreover able to transport DHFR into the lumen with methotrexate bound in the active site, demonstrating that the DeltapH-driven transport of large, native structures is possible by this pathway. However, correct folding is not a prerequisite for transport. Truncated, malfolded DHFR can be translocated by this system, as can physiological substrates that are severely malfolded by the incorporation of amino acid analogs.
Asunto(s)
Proteínas Portadoras/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Plantas/metabolismo , Pliegue de Proteína , Aminopterina/farmacología , Transporte Biológico/efectos de los fármacos , Compartimento Celular , Antagonistas del Ácido Fólico/farmacología , Proteínas de la Membrana/metabolismo , Metotrexato/farmacología , Pisum sativum , Proteínas de Plantas/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/metabolismo , Fuerza Protón-Motriz , Proteínas Recombinantes/metabolismo , Canales de Translocación SEC , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec- or delta pH-dependent pathway. A twin-arginine motif is essential but not sufficient to direct delta pH-dependent targeting; here we show that a second determinant is located in the hydrophobic region. A highly hydrophobic amino acid is found either two or three residues C-terminal to the twin-arginine in all known transfer peptides for the delta pH-dependent system, and substitution of this residue in the 23-kDa (23K) peptide markedly inhibits translocation. Further, whereas the insertion of twin-arginine in a Sec-dependent precursor does not permit efficient delta pH-dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient delta pH-dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective. We conclude that the core signal for this pathway is a twin-arginine together with an adjacent hydrophobic determinant.
Asunto(s)
Arginina/metabolismo , Proteínas de la Membrana/metabolismo , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Transporte Biológico , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutagénesis , Señales de Clasificación de Proteína/químicaRESUMEN
The assembly of the photosynthetic apparatus requires the import of numerous cytosolically synthesised proteins and their correct targeting into or across the thylakoid membrane. Biochemical and genetic studies have revealed the operation of several targeting pathways for these proteins, some of which are used for thylakoid lumen proteins whereas others are utilised by membrane proteins. Some pathways can be traced back to the prokaryotic ancestors of chloroplasts but at least one pathway appears to have arisen in response to the transfer of genes from the organelle to the nucleus. In this article we review recent findings in this field that point to the operation of a mechanistically unique protein translocase in both plastids and bacteria, and we discuss emerging data that reconcile the remarkable variety of targeting pathways with the natures of the substrate precursor proteins.