title: A novel motif in the NaTrxh N-terminus promotes its secretion, whereas the C-terminus participates in its interaction with S-RNase in vitro.
abstract: BACKGROUND
  NaTrxh, 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Avila-Castañeda
authors.forename: Javier Andrés
authors.initials: JA
authors.lastname: Juárez-Díaz
authors.forename: Rogelio
authors.initials: R
authors.lastname: Rodríguez-Sotres
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Bravo-Alberto
authors.forename: Claudia Patricia
authors.initials: CP
authors.lastname: Ibarra-Sánchez
authors.forename: Alejandra
authors.initials: A
authors.lastname: Zavala-Castillo
authors.forename: Yuridia
authors.initials: Y
authors.lastname: Cruz-Zamora
authors.forename: León P
authors.initials: LP
authors.lastname: Martínez-Castilla
authors.forename: Judith
authors.initials: J
authors.lastname: Márquez-Guzmán
authors.affiliation: Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México 04510, Distrito Federal, México. fcg@unam.mx.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cruz-García
mesh_terms: D020816:Amino Acid Motifs; D002473:Cell Wall / Q000378:metabolism / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012260:Ribonucleases / Q000378:metabolism*; D055571:Secretory Pathway; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*; D014026:Nicotiana / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D012260:Ribonucleases