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Pasos clave en el ERAD de proteínas luminales ER reconstituidas con componentes purificados

Alexander Stein¹, Annamaria Ruggiano², Pedro Carvalho²

¹Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

|September 13, 2014

Resumen

Este estudio revela cómo las proteínas ER mal plegadas se exportan al citosol para su degradación. La ligasa Hrd1p forma un conducto, con Cdc48p que extrae las proteínas ubiquitinadas para el procesamiento proteasomal.

Área de la Ciencia:

Biología celular Biología celular.
La degradación de las proteínas.
El estrés del retículo endoplasmático por estrés del retículo endoplasmático

Sus antecedentes:

Las proteínas mal plegadas del retículo endoplasmático (ER) están dirigidas a la degradación a través de la degradación de proteínas asociadas a ER (ERAD).
ERAD implica la retrotranslocación en el citosol, la poliubiquitinación y la degradación proteasomal.
Los mecanismos moleculares precisos de ERAD, en particular para sustratos luminosos (ERAD-L), siguen siendo incompletamente entendidos.

Objetivo del estudio:

Para dilucidar el mecanismo de la degradación de proteínas asociadas a ER de sustratos luminales (ERAD-L) utilizando componentes purificados.
Para caracterizar el papel de la ubiquitina ligasa Hrd1p en el reconocimiento del sustrato y la retrotranslocación.
Para delinear la función del complejo ATPasa Cdc48p y la enzima deubiquitinante Otu1p en la vía ERAD.

Principales métodos:

Reconstitución de la retrotranslocación de ERAD-L utilizando proteínas purificadas de Saccharomyces cerevisiae.
Pruebas bioquímicas para analizar las interacciones proteína-proteína y las actividades enzimáticas.
Utilizando proteoliposomas reconstituidos para estudiar la extracción por membrana de proteínas poliubiquitinadas.

Principales resultados:

Hrd1p, la única proteína de membrana requerida, interactúa con sustratos mal plegados a través de su dominio que abarca la membrana, distinguiéndolos de las proteínas plegadas.
Tanto Hrd1p como el sustrato se someten a poliubiquitinación, lo que facilita el reclutamiento del complejo ATPasa Cdc48p.
La hidrólisis de ATP mediada por Cdc48p libera el sustrato de Hrd1p, y Otu1p recorta las cadenas de ubiquitina tras el reclutamiento y la activación de Cdc48p.

Conclusiones:

Hrd1p actúa como un conducto de membrana, facilitando la retrotranslocación de proteínas mal plegadas desde el lumen ER.
La acción concertada de Hrd1p, Cdc48p y Otu1p impulsa la eficiente extracción y degradación de las proteínas ER mal plegadas.
Este estudio proporciona un modelo mecanicista para ERAD-L, destacando el papel central de Hrd1p en la selección y translocación del sustrato.

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