Abstract
Plant cells are characterized with their central vacuole, an acidic organelle involved in many physiological functions. The acidification of this organelle is based on the existence of two proton pumps, V-ATPase and V-PPase. V-ATPase uses ATP as its energy source to pump proton across vacuole membrane, and V-PPase uses the power generated by PPi hydrolysis to drive the same reaction. The proton gradient established can then be utilized for many secondary transporters.V-PPase contains some highly conserved motifs, one is at Loop 5 region which comprises a eleven-residue sequence “(D/E)X7KXE”. This motif is believed to be involved in PPi binding and hydrolysis mechanism. Transmembrane domain Ⅴ connects the N-terminal region of Loop 5, and many residues within this α-helix are also conserved among species. Hydropathy plot showed that the charged and polar side chains all group around on one side of the outer surface of thisα-helix. It was suggested that this transmembrane domain could be involved in proton translocation mechanism. Recently, in our previous study, it was shown that C-terminal part of V-PPase is important in enzymatic activities.Based on these evidences, we used mung bean (Vigna radiata) V-PPase as our material and created some site-directed alanine mutants to see whether they affect the enzymatic activities of V-PPase. We constructed 6 alanine mutants within transmembrane domain Ⅴ (L223A, I227A, Y230A, L232A, L239A, F240A), one lysine-to-alanine mutant (K261A) located in the motif “(D/E)X7KXE”, and 3 lysine-to-alanine mutants (K624A, K711A, K717A) which are at C-terminal loop region.Four lysine-to-alanine mutants (K261A, K624A, K711A, K717A) went through yeast expression system. The mutant V-PPases were expressed on yeast microsomes and assayed for their enzymatic activities. We found that K261A mutation abolished all V-PPase activities; while the other 3 lysine-to-alanine mutants (K624A, K711A, K717A) located at C-terminal loop region all showed enhanced abilities in PPi hydrolysis and proton pumping. In comparison to other mutants generated previously, in which most have lost some extent of enzymatic activities, it is interesting that we have promoted the abilities of this enzyme by mutating some residues in C-terminal loop region. Again, such data confirm the importance of C-terminal region in V-PPase.Abstract in Chinese--------------------------------------------3Abstract-------------------------------------------------------5Introduction---------------------------------------------------7Materials and Methods-----------------------------------------20Results-------------------------------------------------------30Discussion----------------------------------------------------35References----------------------------------------------------37Tables and Figures--------------------------------------------48