An investigation into the Nicotiana benthamiana XIP1;1 N-terminal region

Abstract

Major intrinsic proteins (MIPs) are proteins that allow the passive transfer of uncharged molecules through a membrane. They have many subfamilies the least understood of which is “X” intrinsic proteins (XIPs). The X illustrates that they are functionally uncharacterized. XIPs have previously been shown to facilitate the transport of boric acid, hydrogen peroxide, urea and glycerol across the membrane. This project focused on the Nicotiana benthamiana XIP1;1. It contains an extraordinary long N-terminal region that previous studies show abolish the expression when truncated. The N-terminal region also contains a lot of serine and threonine residues. These residues are most commonly associated with phosphorylation, which is a post translational modification (PTM). This study created constructs of varying length that would eliminate some of these putative phosphorylation regions and potentially influence the expression of the construct in the expression host Pichia pastoris. P. pastoris is a eukaryote and therefore expected to carry out PTMs similar to higher organisms. Its alcohol oxidase 1 promoter can be induced with methanol to produce high quantities of a specific protein. The constructs were successfully overexpressed by this promoter in P. pastoris and were also shown to be sensitive to boric acid as shown in the boric acid growth toxicity assay. All constructs showed decreased growth when compared to the empty plasmid which indicates that the constructs were successfully translated and inserted into the plasma membrane of the yeast. Another conclusion was that the better performing transformants were found on selection plates with the highest zeocin concentration used. These would have incorporated a high gene copy number of the zeocin resistance selection marker and the construct into the P. pastoris genome, and results indicate that this increases protein expression.

Popular Abstract

Water is the molecule of life. Every cell in our body needs the right water content to function properly. For this to occur water needs a way to enter or exit the cells. One major way for this to happen is by allowing water to pass through proteins that form channels for water in the membrane that delimits the cell. Specific membrane proteins called aquaporins or major intrinsic protein (MIPs) provide for this function. There are many different types of MIPs and they all share a structurally conserved architecture. However not all MIPs facilitate the transfer of water through the membrane. The protein I studied, XIP1;1 from the tobacco plant Nicotiana benthamiana, belong to the “X” intrinsic proteins (XIPs), a subfamily of MIPs, and does not facilitate the transfer of water. The X in XIPs means its function in plants is not known. The XIP selectivity filter is different to MIPs that transport water and previous studies have shown that XIPs can facilitate the transfer of boric acid, hydrogen peroxide, urea and other hydrophobic solutes across the membrane.
A unique aspect of XIP1;1 is that it has a very long N-terminal region. This region is at the beginning of the protein and reside inside the cell. In earlier studies where almost all of this region has been deleted the production of the protein was much reduced. In the N-terminal region there are a lot of sites that could potentially be modified by the addition of a phosphate group. These modifications may structurally change a protein and thereby regulate its localization and function. The aim in this project was to create constructs of various lengths and to investigate the impact of these N-terminal deletions on the expression and function of XIP1;1 protein in the yeast Pichia pastoris. P. pastoris was used because it can be manipulated to produce a high quantity of a specific protein and this has successfully been employed for production of membrane proteins from both plants and animals. Another reason for using this yeast is because it is a eukaryote and it may therefore support similar additions of phosphate groups that potentially occur in plants.
Once the correct constructs in P. pastoris were obtained they were tested, using a boric acid growth toxicity assay. Boric acid was used because it was seen in previous experiments to have a detrimental effect on the growth of yeast cells making closely related XIPs. The slower the growth, the more efficient transfer of boric acid across the membrane. Indeed, each construct grew at a different pace but they all grew slower than a control with no XIP gene. The results showed that the constructs were successfully produced and inserted into the yeast cell membrane. In contrast to yeast, plants need boron to sustain the integrity of the cell wall and boron deficiency is harmful to plants. A plausible physiological function of XIPs is to facilitate the uptake and transport of boric acid in plants.

• author: Leavy, Sinead
• supervisor: Henry Ampah-Korsah
• organization: Department of Chemistry
• course: KEMT30 20151
• year: 2015
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: proteinvetenskap, XIP, major intrinsic protein, Nicotiana benthamiana, Boric acid, Pichia pastoris, membrane, MIP, protein science
• language: English
• id: 7444716
• date added to LUP: 2015-07-02 14:48:34
• date last changed: 2015-07-02 14:48:34