Functional characterization of p24δ subfamily proteins in a. Thaliana
- Autores: Noelia Pastor Cantizano
- Directores de la Tesis: Fernando Aniento Company (dir. tes.), María Jesús Marcote (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2016
- Idioma: español
- Tribunal Calificador de la Tesis: Jean Gruenberg (presid.), Carmen González Bosch (secret.), Alejandro R. Ferrando Monleon (voc.)
- Programa de doctorado: Programa Oficial de Doctorado en Biotecnología
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
p24 proteins constitute a family of small (~ 24kDa) type-I transmembrane proteins which localize to the compartments of the early secretory pathway, including coated protein (COP) I- and COPII-coated vesicles, which mediate the bidirectional transport between the ER and the Golgi apparatus. Based on sequence homology, p24 proteins can be classified into four subfamilies: p24α, p24β, p24γ and p24δ. In contrast to animals and fungi, in plants there are only members of p24β and p24δ subfamilies. In particular, Arabidopsis contains 9 members of the p24δ subfamily which can be divided into two subclasses, δ-1 (p24δ3-p24δ6) and δ-2 (p24δ7-p24δ11), and 2 members of the p24β subfamily. Since p24 proteins cycle between the ER and the Golgi apparatus, they have been proposed to function as putative cargo receptors and to be involved in quality control during protein transport in the early secretory pathway, the organization of ER export sites or the biogenesis and maintenance of the Golgi apparatus. However, their functions in plants are essentially unknown.
The main aim of this work has been the functional characterization of p24 proteins of the p24δ subfamily in Arabidopsis. To this end, two different approaches have been followed.
On the one hand, quadruple knockout (KO) mutants of both p24δ-1 (p24δ3δ4δ5δ6 mutant) and p24δ-2 (p24δ7δ8δ9δ10 mutant) subclasses were obtained. The p24δ3δ4δ5δ6 and p24δ7δ8δ9δ10 mutant did not show phenotypic alterations when they were grown under standard growth conditions, suggesting that p24 proteins from the delta subfamily are not necessary for growth under these conditions in Arabidopsis. The analysis of the levels of p24 proteins in both quadruple mutants showed a decrease in the levels of other p24 proteins, which may be due to a decrease in protein stability and not to a decrease in mRNA levels, suggesting that these p24 proteins may function together in heteromeric complexes. In addition, a possible alteration in the compartments of the early secretory pathway was investigated. It was found that loss of p24δ-1 or p24δ-2 proteins produce alterations mainly in the Golgi apparatus. These data suggest that p24 proteins of the delta subfamily are involved in the maintenance of the structure and organization of the compartments of the early secretory pathway in Arabidopsis. Moreover, the effect of the loss of p24δ proteins in the transport of the K/HDEL receptor ERD2 and a K/HDEL ligand, the chaperone BiP, was also investigated. It was found that loss of p24δ proteins induced the accumulation of the K/HDEL receptor ERD2a-YFP at the Golgi apparatus. This effect was reversed by co-expression of either p24δ5 (δ-1 subclass) or p24δ9 (δ-2 subclass) or of a K/HDEL ligand. In addition, loss of p24δ proteins induced secretion of BiP, probably due to an inhibition of COPI-dependent retrograde Golgi-to-ER transport of ERD2 and, in consequence, in the retrieval of K/HDEL ligands. Finally, it was found that both p24 quadruple mutants show a constitutive activation of the UPR pathway, which may act as a compensatory mechanism that helps the plant to cope with the transport defects in the absence of p24 proteins. Indeed, an increase of the membrane association of several COPI and COPII subunits was observed in both quadruple mutants. In addition, loss of p24δ-1 and p24δ-2 proteins produced the up-regulation of SEC31A, a gene involved in the formation of COPII vesicles.
On the other hand, it was investigated whether Arabidopsis p24 proteins from the delta-1 subclass are glycosylated and if this glycosylation may have functional implications, in particular with respect to their role in sorting ERD2 within COPI vesicles for retrieval of K/HDEL ligands from the Golgi apparatus to the endoplasmic reticulum. It was found that a member of the p24δ-1 subclass, p24δ5, is N-glycosylated in its GOLD domain, in contrast to p24δ9, a member of the p24δ-2 subclass. The N-glycosylation of p24δ5 is not required for its steady-state localization at the endoplasmic reticulum, but it is important for its interaction with the K/HDEL receptor ERD2a and for retrograde transport of ERD2a and K/HDEL ligands from the Golgi apparatus to the endoplasmic reticulum.
