Molecular Characterization Of NtPPME1 Functional Units In Pollen Tube

The unconventional protein secretory is a useful supplement to the conventional protein secretion pathway,It plays an important role in biological function and significance in the life activities and function of the cells.However,the molecular biological mechanism of unconventional protein secretion is unclear.Nicotina tabacum pollen pectin methylesterase 1(Nt PPME1)is the key enzyme regulating cell wall formation.It participates in an unconventional exocytic pathway.Firstly,it is synthesized in endoplasmic reticulum,modified and sorted in Golgi,and transferred directly through Golgi-derived secretory vesicles to apoplast.However,the sorting and exocytic mechanism of Nt PPME1 is unknown.In this study,we employed bioinformatics to predict the structure of Nt PPME1 which includes secondary structure and higher structure,as well as potential glycosylation sites.Thereafter,we further studied the sorting and transporting signals which regulate the intracellular trafficking of Nt PPME1.The subcellular localization and exocytosis secretion pathway were studied by protein truncation and point mutations.We identified that some functional domains of Nt PPME1 can affect unconventional exocytosis pathway:(1)In the process of polar exocytosis of Nt PPME1,both Pro region and PME domain are required.The deletion of any domain could inhibit the Nt PPME1 correct targeting.(2)The Random coil sequence of Nt PPME1 contains the endoplasmic reticulum export signal in Pro region that assists protein transport to cis-Golgi.The modification of random coil will cause the protein to stay in the lumen of endoplasmic reticulum,and further point mutation identified that the four critical amino acids FV and LL played a key role in trafficking from endoplasmic reticulum to Golgi.(3)The C-terminal of Nt PPME1 is plays a key role in trafficking to the apoplast.We have identified 5 amino acids in the C-terminal of Nt PPME1 contribute to the polar targeting of Nt PPME1 targeting of plasma membrane.Further point mutation shows that the entire functional sequence of EAGMM plays an important role in this process.(4)The fourth N-glycosylation site affected the exocytic fusion of Golgi-derived secretory vesicles with plasma membrane.There are four predicted glycosylation sites in Nt PPME1,however,we found the fourth glycosylation site(N482)determined polar trafficking of Nt PPME1,and mutation of this amino acid alone could block the exocytosis process.Further co-localization with the endomembrane system organelles maker showed that most of the proteins stayed in the Golgi-derived secretory vesicles.In conclusion,we employed truncation and point mutation to identify the critical signal that determines the subcellular location and exocytic trafficking of Nt PPME1.It helps to reveal the underlying of unconventional secretion of Nt PPME1 in plant polar cell growth.It provides an important theoretical basis for further exploring and understanding the biological function and mechanism of intracellular unconventional protein secretion pathway.