Regulation of Arabidopsis growth and development by the S-acyltransferase TIP1.

We are finding out how plants grow. One protein, TIP1, is particularly important, and if it isn't working properly plants are dwarfed and end up about a third smaller than normal. We know that TIP1 works by attaching fatty acids to proteins, but we don't know which proteins, or how they affect growth. We will use three methods to find proteins to which TIP1 normally attaches fatty acids (TIP1 targets). First, we will compare proteins with fatty acids attached in normal plants with those in plants that don't have TIP1. Any proteins that only have fatty acids attached when TIP1 is present are likely to be targets of TIP1. Secondly, we will take advantage of the fact that proteins with fatty acids gather in cell membranes. We will compare proteins in the membranes of plant cells grown with and without TIP1 to find out which proteins are only present in membranes when TIP1 is working. Finally, we will use specialised yeast cells that change colour when TIP1 binds to another protein to find plant proteins that bind TIP1. To boost our knowledge of how TIP1 works, we will find out where TIP1 is in plant cells, and find out more about the way that TIP1 attaches fatty acids to proteins. At the end of the project we expect to have identified a lot of TIP1 target proteins, to know whether or not these proteins are normally found in plant cell membranes, and to have a good idea how some of them might contribute to plant cell growth.

The TIP1 gene plays a central role in plant growth and development. Tip1- mutants are dwarves with reduced cell size throughout the plant, and defective root hair and pollen tube growth. We have recently shown that TIP1 encodes the first S-acyl transferase to be identified in plants. S-acylation plays a prominent role in targeting proteins, including trafficking and signaling molecules, to cell membranes, but very few S-acyltransferases have been studied in detail. To understand how TIP1 controls plant growth we will 1. Use pulldown of acylated proteins by biotin substitution of acyl groups, 2D-DIGE comparison of wild type and Tip1- membranes, a yeast 2-hybrid screen, and the phenotypes of existing Tip1- mutants, existing TIP1 over-expression lines, and publicly available insertion lines to identify substrates of TIP1 that affect plant growth and development. 2. Use fluorescence microscopy and TEM immunogold of existing transgenic plants expressing functional proTIP1:TIP1:GFP and proTIP1:TIP1:6HIS:FLAG to discover where TIP1 is localized in plant cells. 3. Use the known TIP1 target ROP10 in acylation assays in vitro and in vivo in Arabidopsis and yeast to characterize the biochemical mechanism of S-acylation.