RESUMEN

The experimental problems associated with in vivo studies of essential proteins or integral membrane proteins have triggered geneticists to generate novel approaches that have often led to insights of general relevance (Shuman and Silhavy, 2003). In order to extend the experimental portfolio, we developed target-directed proteolysis (TDP), an in vivo method allowing structural and functional characterization of target proteins in living cells. TDP is based on the activity of the highly sequence-specific NIa protease from tobacco etch virus. When its recognition site of seven residues is engineered into target proteins and NIa protease is expressed under tight promoter control, substrates can be conditionally processed while other cellular proteins remain unaffected. Applications include conditional inactivation as well as functional characterization of target proteins.

Asunto(s)

Endopeptidasas/metabolismo , Proteínas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Endopeptidasas/genética , Hidrólisis , Proteínas/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Target directed proteolysis allows specific processing of proteins in vivo. This method uses tobacco etch virus (TEV) NIa protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed.

Asunto(s)

Endopeptidasas/metabolismo , Ribosomas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Citoplasma/metabolismo , Endopeptidasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Translocación SEC , Proteína SecA , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismo