Investigations of the role of peroxisomes in sterol biosynthesis in the slime mould Dictyostelium discoideum

Dictyostelium discoideum is a good model organism to study a variety of cellular processes. Its complete genome sequence is known (dictybase.org). It has a unique life style, with motile unicellular and multicellular stages, and multiple cell types (Annesley and Fisher 2009). It is widely used to study chemotactic motility and cytoskeletal dynamics. It is also used for studying molecular pathogenesis and treatment of human disease. Furthermore, since Dictyostelium cells readily take up drugs, it has led to identification of drug targets. For instance, farnesyl diphosphate synthase was found, by use of D. discoideum, to be the primary target of bisphosphonate drugs that are widely used to prevent and treat osteoporosis.
A major challenge with this organism is the difficulty of making targeted mutations by homologous recombination. It is difficult to generate targeting constructs because of the high AT contents (77.4%) of its genome. Introduction of the Clustered Regularly Interspaced Short Palindromic Repeats system (CRISPR) into D. discoideum for obtaining gene knockouts would therefore be potentially most helpful and here I report on our progress in setting this up.
All previous investigations of the intracellular location of the first four enzymes involved in sterol biosynthesis from farnesyl diphosphate (FDP) have found that they locate to the ER membrane in eukaryotic cells. However, we provide strong evidence that these enzymes are peroxisomal in D. discoideum. The first step in the pathway is catalysed by squalene synthase SQS. Surprisingly, (DdSQS) contains a typical peroxisomal targeting signal type 1 (PTS1). All known PTS1-containing proteins are localized to the peroxisomal matrix whereas membrane proteins use a different pathway. However, we found that DdSQS behaves as a membrane protein. Interestingly, as in other organisms, the DdSQS has a C-terminal amino acid sequence potentially forming a hydrophobic helix but which in D. discoideum is located immediately upstream of the PTS1. Deletion of this helix does not affect peroxisomal targeting but does affect DdSQS association with the membrane and may therefore serve as a tail anchor. Furthermore, the helix plays an important role in forming a SQS homodimer. SQS is the first example of a peroxisomal membrane protein that makes use of the PTS1 pathway for its localization.

We have shown that the first four enzymes of sterol biosynthesis from FDP are associated with the peroxisomal membrane by using structured illumination microscopy. This is a relatively new technique that allows imaging beyond the resolution limit for light microscopy. Furthermore, the topology of the enzymes with respect to the peroxisomal membrane was experimentally determined. We provide a model of how sterol biosynthesis from FDP takes place in the peroxisomes of D. discoideum.