Universität Tübingen
Identification of a putative SUT1 mRNA binding protein using yeast three-hybrid analysis
Abstract
Sucrose is the major nutrient product of the photosynthesis reactions; its correct distribution via the specialized cells of the phloem is essential to plant survival. The transport of sucrose within solanaceous plants is mediated in part by sucrose transport proteins (SUTs) localized to the plasma membrane of the phloem sieve elements (SEs). SUT1, which is essential for phloem loading of sucrose, shows the predicted localization to the enucleate SE; its mRNA, initially thought to be found exclusively in the closely associated companion cell (CC), is also found in the enucleate SE. The mechanism of arrival and purpose of this mRNA localization in solanaceous plants has become a matter of dispute. The present work deals with the application of an in vivo method for the discovery of mRNA binding proteins, and the identification of a putative LeSUT1 mRNA binding protein. A high quality cDNA library encoding tomato leaf protein fusions was synthesized and cloned for use in the yeast three-hybrid method (SenGupta et al., 1996). Several RNA hybrid molecules were designed based on the sequence and secondary structure of LeSUT1 mRNA and used to screen the cDNA library for putative mRNA binding proteins. A single isolated cDNA clone was shown to interact with two structurally dissimilar RNA baits. Northern blot and RACE analysis suggest that the isolated cDNA is a fragment of a larger molecule; sequence analysis suggests the presence of a putative RNA binding domain. The isolated cDNA encodes a novel protein molecule that is able to specifically interact with LeSUT1 mRNA in vivo and may be involved in the localization of SUT1 mRNA. While the localization of SUT1 protein was previously established (Kühn et al., 1997), the mechanism of this localization has yet to be identified. In order to examine this process, tobacco sections were exposed to the fungal toxin Brefeldin A (BFA), which inhibits anterograde vesicle transport of proteins. SUT1 protein localization was inhibited by BFA application to fresh sections in preliminary experiments. When fresh tobacco sections were incubated in water, or exposed to no treatment, SUT1 protein was predictably localized to the SE. When fresh sections were exposed to 50 mM BFA for 10 minutes, however, SUT1 protein was sequestered in the CC; was visibly increased upon exposure to 100 mM BFA. These initial results contribute to the formation of an emerging model of SUT1 localization in which protein and mRNA are localized in separate but related events. The highly determined secondary structure of LeSUT1 3'UTR as well as the existence of multiple polyadenylation states also serves to support the concept of a highly regulated complex system resulting in the localization of SUT1 to the plasma membrane of the enucleate SE.
Author and committee
dc:creator, dc:contributor.*- Author
-
- Panford-Walsh, Rama N.
Identifiers
dc:identifier.*- Identifier
- hdl:10900/48575