RESUMEN
Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coli auxotroph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides. In vitro protein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant "mature" spinach lumazine synthase is expressed in E. coli at levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step procedure. Apparent V(max) and K(m) values obtained with the purified plant protein are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E. coli and Bacillus subtilis.
Asunto(s)
Cloroplastos/enzimología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Riboflavina/biosíntesis , Spinacia oleracea/enzimología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Bacterias/enzimología , Secuencia de Bases , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Prueba de Complementación Genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Plantas Tóxicas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Spinacia oleracea/genética , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.
Asunto(s)
Transferasas Alquil y Aril , Genes de Plantas , Ligasas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloroplastos/enzimología , Clonación Molecular , Frutas , Regulación Enzimológica de la Expresión Génica , Prueba de Complementación Genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fenómenos Fisiológicos de las Plantas , Plantas/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.