A short motif sequence that might help to predict if the protein is secreted or associated to the plasmatic membrane

Andre Zaragoza-Gómez1, James González-Flores2, Víctor Hugo Anaya-Muñoz3, Felipe Cruz-García4, Javier Andrés Juárez-Díaz1*
1. Departamento de Biología Comparada, Facultad de Ciencias, UNAM; 2. Departamento de Biología Celular, Facultad de Ciencias, UNAM; 3. ENES-Morelia, UNAM; 4. Departamento de Bioquímica, Facultad de Química, UNAM.
*Corresponding author/Speaker (j.a.juarezdiaz@ciencias.unam.mx)

Summary Results
NaTrxh is a thioredoxin type h (subgroup 2) from Nicotiana alata recently characterized as an essential protein in gametophytic S-RNase-based self- BLAST analysis using the sequence of the Nβ motif resulted in a primary list that was filtered by deleting synonim of the proteins that
incompatibility system. A peculiar feature of NaTrxh is that although it lacks a signal peptide, it localizes into the extracellular matrix of the stylar transmitting contain it (14). So far, we have analyzed the sequences belonging to eukaryotes (15).
tract of N. alata. Transient expression of NaTrxh-GFP in leaves (N. benthamiana and Arabidopsis thaliana) and in onion epidermal cells show that the
information to lead its secretion is contained in the sequence of NaTrxh itself. This information is within a motif formed by 11 amino acids, called Nβ, which
in N. alata correspond to Ala-17 to Pro-27.
In order to determine if the Nβ motif might function as a signal/transit peptide-like sequence in proteins that despite lacking any of a typical signal/transit
peptide, their localization is extracellular, we searched proteins with this motif (or a similar one). Then, from the obtained sequences, the synonym proteins
(same sequence, different accession number) were eliminated, and the rest were analyzed according to the source, function, and cellular localization. The
position in which the Nβ motif was located within each primary structure was also analyzed (all sequences were divided in four quarters).
Preliminary data suggest that when the Nβ motif is located in the first quarter of the protein (and maybe the second one as well), most of the proteins
appear to be secreted or membrane-associated. Apparently, none of these proteins contain a signal or transit peptide suggesting a correlation between the
presence of the Nβ motif and a particular localization (membrane or secreted).
Overall, our data suggest that determining the presence of the Nβ motif and its position within the sequence, might be used as a tool to predict a putative
cellular localization of the proteins that contain it, indicating that this motif is a novel signal/transit peptide.

Background Total 383 14 Total 306 15

Transient expression assays of NaTrxh-GFP in onion epidermal cells (1) and in leaves of A. thaliana (2) and N. benthamiana (3), indicate that NaTrxh
possesses the information to lead its secretion. The NaTrxh N-terminal contains the responsible motif for its extracellular localisation: the Nβ motif (Ala-17 Subsequently, the eukaryotic sequences were clustered according to the position in which Nβ motif was located within the primary structure.
to Pro-27) (4-9), which contains atypical features as a signal peptide. The location of the Nβ motif within the primary structure of NaTrxh (4) and its For this, the sequences were splited into four equal parts (16), and then, were classified regarding the reported function (17-20).
hydrophilic profile suggested a protein secretion pathway independent or alternative to the ER and Golgi apparatus. However, cellular evidences show that
it does utilize them (10-13). This indicates that the Nβ motif represents a novel signal directing protein secretion in cells. Position 1 Position 2
a
1 4 Nα motif NaTrxh core
Ribosome

Onion epidermal cells

Nβ motif Active site COO motif SRP ?+ mRNA

b 5 a
2 NaTrxh-GFP:KDEL
8 a 10 16
Primary structure
NaTrxh∆Nα-GFP
6 a 1/4 2/4 3/4 4/4
Position 1 Position 2 Position 3 Position 4
17 18
NaTrxh∆Nβ-GFP NaTrxh-GFP + Brefeldin A Total 106 Total 65
11 Position 3 Position 4
9 a
A. thaliana leaf cells
NaTrxh∆Nαβ-GFP
b
3 7 a

Nβ-GFP NaTrxh immunolocalization

12
NaTrxh∆COO-GFP

N. benthamiana leaf cells
13
Modified from:
a Ávila-Castañeda, A., Juárez-Díaz, J., Rodríguez-Sotres, R., Bravo-Alberto, C., Ibarra-Sánchez, C.,
Zavala-Castillo, A., Cruz-Zamora, Y., Martínez-Castilla, L., Márquez-Guzmán, J., Cruz-García, F.
(2014) A novel motif in the NaTrxhN-terminus promotes its secretion, whereas the C-terminus
participates in its interaction with S-RNase in vitro. BMC Plant Biol. 14:147
19 20
b Juárez-Díaz, J. A., McClure, B., Vázquez-Santana, S., Guevara-García, A., León-Mejía, P., Total 45 Total 87
Márquez-Guzmán, J., Cruz-García, F. (2006) A novel thioredoxin h is secreted in Nicotiana alata and
reduces S-RNase in vitro. J. Biol. Chem. 281: 3418-3424.

DGAPA-UNAM IA204815 and IN230920

CONACyT 240927