Compartmentalisation of Mevalonate Biosynthesis in Evolutionarily Divergent Eukaryotes

Mevalonate is a committed precursor of sterols and other isoprenoids in mammals, yeast, plants and many other eukaryotes. A key enzyme in the mevalonate pathway of isoprenoid biosynthesis is HMG-CoA reductase (HMGR), which represents a key regulatory step in mammals and potentially other organisms, responsible for the reduction of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA). The classical localisation of this enzyme is within the endoplasmic reticulum (ER) as an integral membrane protein with a cytosolic facing enzymatic domain. However, the trypanosomatid HMGR does not fit this description. It is instead localised within the mitochondrion. There is also indication from genome analyses that in a variety of other microbial eukaryotes HMGR may be present as soluble, rather than integral membrane protein, but where within cells these in silico-identified HMGRs localise is not known. The mitochondrial localisation of HMGR in trypanosomatids may be an adaption to allow the amino acid leucine to be utilised as a major carbon source for sterol biosynthesis. Leucine breakdown occurs within mitochondria in eukaryotes and results in HMG-CoA as an intermediate however, due to the organellar separation of these processes in most eukaryotes leucine cannot be utilised as in trypanosomatids.

In order to investigate a possible adaption to the mevalonate pathway in different divergent eukaryotes, two organisms and two genes were selected. Squalene epoxidase (SqE), an enzyme near the end of the pre-squalene section of the mevalonate pathway was investigated in the trypanosomatid Leishmania tarentolae. The localisation of this enzyme was not clear form past studies and literature comparisons, being possibly peroxisomal, ER or lipid-body localised. Homologous recombination via a two-step PCR originated tagging amplicon was utilised, resulting in an N-terminal green fluorescent protein (GFP) tag for the SqE gene in L. tarentolae cells. These cells were then analysed using confocal microscopy, and although the homologous recombination appeared to be successful, the imaging data was too unclear as to provide accurate localisation information.

Dictyostelium discoideum was the major organism of focus for this study. This is an early diverging eukaryote, realistically sharing its last common ancestor with the trypanosomes at the very root of all eukaryotic lineages. The genes of focus in this organism encoded the HMGR isoforms, DdHMGA and DdHMGB. Myc-tagged synthetic genes for both isoforms were cloned into Dictyostelium-specific expression vector pDM1039 and transfected into D. Transformed cells, including those also expressing Red Fluorescent Protein (RFP) targeted to peroxisomes, were analysed by immunoblotting (to size expressed myc-tagged HMGR proteins) and immunofluorescence confocal microscopy. DdHMGA was shown to possess a possible peroxisomal localisation while DdHMGB showed a punctate possible cytosolic localisation.

Alongside the laboratory studies, computational studies were carried out in order to investigate the presence or lack of various mevalonate pathway enzymes and their localisation within a wide range of eukaryotes. In this way I aimed to provide new insight into the organisation and evolution of the important mevalonate pathway in eukaryotes.