Biochemical and structure-function analysis of PIN proteins

Auxin is a major plant hormone that has to be properly transported and redistributed throughout the plant at all stages of a plant`s life cycle. The proteins involved in controlling and directing auxin distribution are thus key determinants of many aspects of plant growth, development and physiological responses. The PINs are a family of integral membrane proteins that play a crucial role in the export of auxin from cells, but the actual mode of action by which they mediate or control auxin transport is unknown. As PINs show no significant homology to other proteins, the lack of experimental data on their molecular properties hampers progress in this field. The proposal aims to address this gap in our knowledge by using biochemical approaches to elucidate some of the basic structural features of PIN proteins, such as determining membrane topology and investigating the occurrence of N- glycosylation as well as inter/intra-molecular disulfide bonds. Another objective is to isolate and characterise oligomeric forms of PINs, as oligomerisation is a feature that is integral to the mechanism of action of many membrane transporters and receptors. The biological significance of any identified structural features will be investigated by analysing their effects on PIN function in vivo. Overall, the data from this project should reveal some of the molecular features of PIN proteins, which will aid in the interpretation of past and future studies on the involvement of PINs in plant auxin distribution.

Plants have a growth hormone called auxin, which masterminds all stages of plant growth and development, from the embryo through to seedling, juvenile, adult, reproductive and senescent stages. Without this potent regulator, plants would not survive, and any pertubations to the normal working of auxin causes growth abnormalities. A hallmark feature of auxin is that it is carefully transported and redistributed through the plant, e.g. from the shoot down to the roots. As this ability to be transported is integral to its function as a hormone, the transport of auxin is an area of intensive research how auxin transport is performed, how it is controlled, what effects it has and the problems that plants face when the transport process goes awry. Since plants (like all living organisms) are composed of cells, auxin transport involves getting the auxin molecules into a cell, then out again and into the next cell, and so on. To determine the particular direction that auxin should go, plants restrict the outward transport so that auxin can only leave at one end of the cell. This is done by limiting the auxin export machinery to one end only. This machinery consists of complicated protein molecules called PINs. This FWF project studied the PINs to try to understand how they work at the molecular level. The results give us an idea of what PIN molecules look like in their native state, how they might transport auxin, how they are organised into large multi-complexes, and how these complexes are made. One of the most interesting findings was that the complexes differed between the three PINs that were investigated (plants have eight PINs, this study looked at three major ones), even when all three were found in the same cell, suggesting that plants may use such differences to control each PIN in a different way. In addition, this project also revealed how the PINs have evolved as transporters and their relationship to other non-auxin transporters, which is very useful as it allows us to understand the PINs by comparing them to other types of well-studied transporters for which more information is available.