FUNCTIONAL CHARACTERIZATION of P24δ SUBFAMILY PROTEINS in A. Thaliana”

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FUNCTIONAL CHARACTERIZATION of P24δ SUBFAMILY PROTEINS in A. Thaliana” UNIVERSITAT DE VALÈNCIA Departamento de Bioquímica y Biología Molecular Programa de doctorado en Biotecnología “FUNCTIONAL CHARACTERIZATION OF p24δ SUBFAMILY PROTEINS IN A. thaliana” Tesis doctoral con Mención Internacional presentada por Noelia Pastor Cantizano Tesis dirigida por: Dr. Fernando Aniento Company Dra. María Jesús Marcote Zaragoza Valencia, Octubre 2016 FERNANDO ANIENTO COMPANY, Catedrático del departamento de Bioquímica y Biología Molecular de la Universidad de Valencia y MARIA JESÚS MARCOTE ZARAGOZA, Profesora titular del departamento de Bioquímica y Biología Molecular de la Universidad de Valencia, CERTIFICAN: Que la presente memoria titulada: “FUNCTIONAL CHARACTERIZATION OF p24δ SUBFAMILY PROTEINS IN A. thaliana” ha sido realizada por la Licenciada en Farmacia Noelia Pastor Cantizano bajo nuestra dirección, y que, habiendo revisado el trabajo, autorizamos su presentación para que sea calificada como tesis doctoral y obtener así el TITULO DE DOCTOR CON MENCIÓN INTERNACIONAL. Y para que conste a los efectos oportunos, se expide la presente certificación en Burjassot, a 19 de octubre de 2016. Fdo: Fernando Aniento Company Fdo: María Jesús Marcote Zaragoza Esta Tesis Doctoral se ha realizado con la financiación de los siguientes proyectos: 1. “Tráfico intracelular de proteínas en células vegetales”. Plan Nacional de I + D. Programa de Biología Fundamental (BFU2009-07039). I.P.: Dr. Fernando Aniento Company. 2. “Tráfico intracelular de proteínas en células vegetales”. Plan Nacional de I + D. Programa de Biología Fundamental (BFU2009-07039). I.P.: Dr. Fernando Aniento Company. 3. “Tráfico intracelular de proteínas en células vegetales” GEVA- Generalitat Valenciana. I.P.: Fernando Aniento Company El presente trabajo de investigación ha sido desarrollado durante el periodo des disfrute de una beca predoctoral FPU (Formación Profesorado Universitario) del Ministerio de Educación (AP2010-2326), durante los años 2009-2013, y una ayuda de la Universidad de Valencia para la realización de Estancias Breves en la “School of Life Sciences” de la Universidad China de Hong Kong. AGRADECIMIENTOS Y por fin llegó el momento de parar un segundo y echar la vista atrás para agradecer a todos los compañeros de viaje que he tenido durante estos años. En primer lugar, me gustaría agradecer a mis directores Fernando y María Jesús su dedicación y su enorme paciencia, además de todo el cariño que me han dado estos años. Gracias también a mi querido Consejo Privado por todos esos buenos momentos dentro y fuera del lab y por todo el apoyo que me han dado incluso desde la distancia: Juan Carlos (obviamente la mano del Rey), gracias por ser mi maestro en el laboratorio; Gloria, te espero allá donde vaya para vivir más aventuras; y Joan, sigue intentando sacar una sonrisa a los demás en cualquier situación. A Fátima y César, a los que seguro que les va a ir muy bien en esto de la ciencia. A Pilar (ya te he dicho que McGyver a tu lado no sabe hacer nada) gracias no solo por haberme facilitado el trabajo sino también por todas esas charlas estos últimos meses. También quiero agradecer a toda la gente del departamento de Bioquímica su cariño durante todos estos años. Y ya puestos a quedarnos en el tercer piso, no me puedo olvidar de mi María, mi compañera de risas, llantos y cervezas, gracias por estar ahí en tantos momentos, sabes que nunca habría llegado al final sin ti. Y no puedo dejarme a Kelly, sin ella Hong Kong no habría sido lo mismo. Una de las cosas que ha cambiado mientras hacía la tesis ha sido mi forma de disfrutar del deporte y eso se lo tengo que agradecer a que he tenido a los mejores monitores y amigos: Sebi, Marga y Pau. Y ahí conocí a dos personas que ahora mismo son muy importantes para mí. Babé, gracias por compartir conmigo tantísimos momentos, por ser capaz de hacer que lo de todo en un combat con un simple “¿preparada?” y por acudir siempre que necesito un abrazo. Sof, siempre serás mi mama pato que cuida de mi, gracias por tratarme siempre como tu hermana pequeña y estar a mi lado. Gracias también al resto de mi querida fila de atrás (Vane y Honga), a L-na, a mi Pili que ha sido mi gran apoyo estos últimos 10 meses y en pocas clases me ha dejado sola, a mis Chos (Ruth y Cristina) que aunque han sido la última incorporación en mi vida ya se han convertido en dos personitas muy importantes (Be junco my friend!), y aún montón de personas que más que me dejo! Gracias también al resto de mis queridos “The Milk”, a Javi, Jorge, Irene y sobre todo a Dani y Merch por esos picnics ilegales, por comer la tarta con las manos y apuntarse a cualquier plan descabellado (por introducir el término merchada en mi vida!). A mis golfos favoritos (Nacho, Cris, Mow, Mari, Carlos, Vane, César, Pablo y Silvia) por todos esos grandes momentos rodeados comida y cerveza. A Damian, por hacer de su casa siempre nuestra casa también. A mis alumnas, Mari y Silvia, por enseñarme ellas a mí. Y no me puedo olvidar de mis estudiants post-aborigens (Inés, Eva, Nona y Ange) con los que empecé en la universidad y aún siguen a mi lado. Pero si hay dos personas que han estado a mi lado casi desde el momento que puse un pie en la universidad han sido Jorge y Tam. Jorge, gracias por estar siempre a mi lado y aguantarme en tantos momentos, ninguna de mis etapas habría sido lo mismo sin ti, sé que estarás en las próximas también. A mi sis, por ser la niña mágica que un día el destino quiso poner en mi camino, aquel día no sabía lo afortunada que fui pero 11 años después te puedo decir que es un placer recorrer este camino a tu lado. Gracias a la familia de Amadeo por tratarme como una más, sobre todo a mi “cuñao” Fran, a mi cuñada, Marisa y a mi suegra, Luisa, porque en el fondo y sin el fondo os quiero muchísimo. No me puedo olvidar de mi familia de Ceuta, especialmente a mi Tita Luchi, a la que quiero como si fuera mi segunda madre, y a los que me cuidan desde el cielo. Dicen que la familia no se elige, pues a la mía la quiero muchísimo con sus defectos y sus virtudes. A mi hermana, Ana, por preocuparse tanto por mí. A mis niños, Alejandro y Adrián, espero que me perdonéis porque mis escapadas para veros no han sido todas las que me hubieran gustado. Sabéis que os quiero muchísimo. A mi hermano Rafa, porque siempre ha sido mi ejemplo a seguir, sin él hoy no estaría aquí y por ser además mi mejor amigo en todo momento. Y a mis padres, por todo el sacrificio que han hecho para que haya podido llegar hasta aquí, por todo su amor y por ser las personas con el corazón más grande y fuerte que conozco. Y por último, a la persona que más ha sufrido esta tesis conmigo, la que ha estado en mis días buenos, en los malos y en mis noches de trabajo todos estos años. Amadeo, sí quiero compartir contigo lo que esté por venir, sí quiero estar a tu lado hasta que seamos abuelitos y echemos la vista atrás y veamos todo el camino que hemos recorrido juntos, sí quiero pasarme el resto de mis días junto a ti. Gracias por apoyarme siempre e impulsarme para que siga adelante. Te quiero. A Rafa, a mis padres y a Amadeo TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENTS ................................................................... 11 ABBREVIATIONS .......................................................................... 19 ABSTRACTS ................................................................................. 21 ABSTRACT ....................................................................................................23 RESUMEN ....................................................................................................27 RESUM ........................................................................................................31 IN TRODUCTION .......................................................................... 35 1. INTRACELLULAR MEMBRANE TRAFFIC .................................. 37 1.1. Membrane trafficking pathways ........................................................37 1.2. Vesicle trafficking ...............................................................................39 1.2.1. Steps of the vesicular transport. ................................................40 1.2.2. Principles of vesicular trafficking ...............................................46 2. THE SECRETORY PATHWAY .................................................. 47 2.1. Early secretory pathway .....................................................................50 2.1.1. The ER network ..........................................................................51 2.1.2. ER export sites and ER-to-Golgi transport .................................53 2.1.3. Golgi-to-ER transport .................................................................59 2.2. Late secretory pathway ......................................................................62 2.2.1. The trans-Golgi Network (TGN)..................................................62 2.2.2. Transport to the plasma membrane ..........................................63 2.2.3. Vacuolar cargo trafficking pathway ...........................................64 2.3. Unconvetional protein secretion (UPS) ..............................................65 11 TABLE OF CONTENTS 3. VESICLE TRAFFICKING MACHINERY AT THE ER-GOLGI INTERFACE ............................................................................... 68 3.1. COPII vesicles ......................................................................................68 3.1.1. Formation of COPII vesicles .......................................................68 3.1.2. COPII interaction motifs
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