A novel motif in the NaTrxh N-terminus promotes its secretion, whereas the C-terminus participates in its interaction with S-RNase in vitro

BMC Plant Biology

Research Article

Javier Andrés Juárez-Díaz¹  Judith Márquez-Guzmán¹  Claudia Patricia Ibarra-Sánchez²  Rogelio Rodríguez-Sotres²  Yuridia Cruz-Zamora²  Alejandra Ávila-Castañeda²  Carlos E Bravo-Alberto²  León P Martínez-Castilla²  Alejandra Zavala-Castillo²  Felipe Cruz-García² 

[1] Departamento de Biología Comparada, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México, Distrito Federal, México;Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México, Distrito Federal, México;

关键词: Thioredoxin;    Secretion;    Self-incompatibility;    Nicotiana alata;    Gametophytic;    S-RNase;   

DOI  :  10.1186/1471-2229-14-147

 received in 2014-01-24, accepted in 2014-05-12,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundNaTrxh, a thioredoxin type h, shows differential expression between self-incompatible and self-compatible Nicotiana species. NaTrxh interacts in vitro with S-RNase and co-localizes with it in the extracellular matrix of the stylar transmitting tissue. NaTrxh contains N- and C-terminal extensions, a feature shared by thioredoxin h proteins of subgroup 2. To ascertain the function of these extensions in NaTrxh secretion and protein-protein interaction, we performed a deletion analysis on NaTrxh and fused the resulting variants to GFP.ResultsWe found an internal domain in the N-terminal extension, called Nβ, that is essential for NaTrxh secretion but is not hydrophobic, a canonical feature of a signal peptide. The lack of hydrophobicity as well as the location of the secretion signal within the NaTrxh primary structure, suggest an unorthodox secretion route for NaTrxh. Notably, we found that the fusion protein NaTrxh-GFP(KDEL) is retained in the endoplasmic reticulum and that treatment of NaTrxh-GFP-expressing cells with Brefeldin A leads to its retention in the Golgi, which indicates that NaTrxh uses, to some extent, the endoplasmic reticulum and Golgi apparatus for secretion. Furthermore, we found that Nβ contributes to NaTrxh tertiary structure stabilization and that the C-terminus functions in the protein-protein interaction with S-RNase.ConclusionsThe extensions contained in NaTrxh sequence have specific functions on the protein. While the C-terminus directly participates in protein-protein interaction, particularly on its interaction with S-RNase in vitro; the N-terminal extension contains two structurally different motifs: Nα and Nβ. Nβ, the inner domain (Ala-17 to Pro-27), is essential and enough to target NaTrxh towards the apoplast. Interestingly, when it was fused to GFP, this protein was also found in the cell wall of the onion cells. Although the biochemical features of the N-terminus suggested a non-classical secretion pathway, our results provided evidence that NaTrxh at least uses the endoplasmic reticulum, Golgi apparatus and also vesicles for secretion. Therefore, the Nβ domain sequence is suggested to be a novel signal peptide.

【 授权许可】

Unknown   
© Ávila-Castañeda et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.