Characterising the regulatory effects of splice variants of transporters

Abstract

Membrane transport proteins, also known as transporters, are crucial for the maintenance of cell physiology by facilitating the movement of ions, nutrients, metabolites, and waste across cell membranes. Transporter function can be expanded through the mechanism of alternative splicing, which can produce distinct protein isoforms from a single gene. A review of the literature has uncovered a subset of naturally occurring splice variants of eukaryotic transporters with striking similarities. The transporters are unrelated and from a wide range of organisms (for instance, the malaria parasite, the honey bee, and humans); yet their splice variants are all predicted to have grossly deformed topologies, are unable to transport the substrate(s) of their full-length transporter, and instead downregulate the expression of their full-length protein.

In this Thesis, the Xenopus laevis oocyte expression system was employed to characterise the regulatory activities of a selection of these transporter splice variants. The results of transport assays and western blot analyses highlighted that the splice variants all exerted similar downregulatory effects on their respective full-length transporter, regardless of the diversity in the proteins studied. Importantly, it was found that the transporter splice variants also downregulated the expression of unrelated transporters. These findings suggested that the splice variants act through a shared regulatory mechanism.

To gain insight into the nature of this potential mechanism, a number of possible modes-of-action of the transporter splice variants were investigated in X. laevis oocytes. Various experimental approaches were used to exclude several pathways from involvement in the regulatory activities of the splice variants; these include degradation of transporter mRNA and general inhibition of protein translation. However, the exact nature of the regulatory mechanism of the splice variants remains unclear.

We theorise that the deformed topologies of the transporter splice variants trigger folding stress in the ER during their synthesis, thus activating pathways that would rapidly modify ER function as well as broader cell physiology. The splice variants may therefore function as part of a cellular response to environmental changes, through a process that is more efficient than the de novo transcription and translation of distinct regulatory proteins. The work presented in this Thesis contributes to our understanding of this subset of seemingly 'non-functional' splice variants of transporters that may mediate a regulatory mechanism conserved across eukaryotes. Show more