Fatty acid synthesis in African trypanosomes: a solution to the myristate mystery.

The glycosyl phosphatidylinositol anchor of the trypanosome variant surface glycoprotein contains myristate as its sole fatty acid component. Surprisingly, there does not appear to be enough myristate in either the parasite or its host's bloodstream to sustain myristoylation of the enormous quantity of variant surface glycoprotein produced. Here, we discuss how the trypanosome solves its myristate dilemma. The parasite not only efficiently salvages and processes myristate from the bloodstream, but it also makes myristate de novo using a recently discovered specialized fatty acid synthesis system.

Fatty acid remodeling of glycosyl phosphatidylinositol anchors in Trypanosoma brucei: incorporation of fatty acids other than myristate.

Trypanosoma brucei is the protozoan parasite that causes African sleeping sickness. Its surface is packed with 10(7) copies of the glycosyl phosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). This GPI anchor is unusual in that it contains two myristates (14:0) in its lipid moiety. This fatty acid specificity is achieved through myristoylation of the GPI precursor, and the acyltransferases involved in the GPI remodeling were presumed to be specific for myristate. However, their specificity had never been fully evaluated. Here we found as expected that the remodeling acyltransferases completely excluded palmitate (16:0) and stearate (18:0) in a cell-free fatty acid remodeling system. In contrast, we found surprisingly that one of these enzymes was permissive to shorter fatty acids such as laurate (12:0) and octanoate (8:0). However, the rates of incorporation of shorter fatty acids were lower than that of myristate at low substrate concentration. Since shorter fatty acids are virtually absent in the parasite and in the host bloodstream, it is unlikely that shorter fatty acids compete effectively with myristate as remodeling substrates under physiological conditions. Even if they were present in small quantities, a recently identified specialized fatty acid synthetase efficiently elongates shorter fatty acids to myristate prior to incorporation into GPIs (Morita et al., Science 288 (2000) 140-3.). Therefore, even though a remodeling acyltransferase is permissive with regard to substrate chain length, the myristate specificity in GPI anchors is very high.

Glycosyl phosphatidylinositol myristoylation in African trypanosomes. New intermediates in the pathway for fatty acid remodeling.

Glycosyl phosphatidylinositol (GPI) anchors in the bloodstream form of Trypanosoma brucei are unusual in that their two fatty acids are myristate. The myristates are added in the final stages of GPI biosynthesis in a remodeling reaction. Remodeling occurs first at the sn-2 position of glycerol, involving removal of a longer fatty acid and subsequent attachment of myristate. The second myristate is then incorporated into the sn-1 position, but the mechanism has been unclear due to the unavailability of a reliable cell-free system supporting complete remodeling. Here, we first refined the cell-free system (by removing Mn(2+) ions), thereby allowing efficient production of the dimyristoylated GPI precursor. Using this improved system, we made three new discoveries concerning the pathway for fatty acid remodeling. First, we discovered a monomyristoylated GPI (known as glycolipid theta') as an intermediate involved in remodeling at the sn-1 position. Second, we found an alternative pathway for production of glycolipid theta, the first lyso intermediate in remodeling. The alternative pathway involves an inositol-acylated GPI known as glycolipid lyso-C'. Finally, we found that there is significant breakdown of GPIs during remodeling in the cell-free system, and we speculate that this breakdown has a regulatory role in GPI biosynthesis.

Specialized fatty acid synthesis in African trypanosomes: myristate for GPI anchors.

African trypanosomes, the cause of sleeping sickness, need massive amounts of myristate to remodel glycosyl phosphatidylinositol (GPI) anchors on their surface glycoproteins. However, it has been believed that the parasite is unable to synthesize any fatty acids, and myristate is not abundant in the hosts' bloodstreams. Thus, it has been unclear how trypanosomes meet their myristate requirement. Here we found that they could indeed synthesize fatty acids. The synthetic pathway was unique in that the major product, myristate, was preferentially incorporated into GPIs and not into other lipids. The antibiotic thiolactomycin inhibited myristate synthesis and killed the parasite, making this pathway a potential chemotherapeutic target.

Localization of Dictyostelium myoB and myoD to filopodia and cell-cell contact sites using isoform-specific antibodies.

To date, five myosin I heavy chain genes (myoA-E) have been sequenced in Dictyostelium. Among them, myoB, myoC and myoD possess tail domains that contain a putative membrane-binding domain, a nucleotide-insensitive actin-binding site, and an src homology (SH)-3 domain. In this study, we have determined the intracellular localizations of myoB and myoD by immunofluorescence using isoform-specific antibodies raised against bacterially expressed fusion proteins. MyoB is concentrated at the leading edges of lamellipodia and at sites of cell-cell contact in both stationary and aggregation stage cells. Based on its distinctive appearance, we have named the myosin I-rich, interdigitating cell-cell contact structure in the stationary stage cells "zipper". To analyze the staining of filopodia, we employed the ratio imaging technique. We find that myoB is present in filopodia in either a uniform or an apical staining pattern. Like myoB, myoD is concentrated in leading edges of lamellipodia and at sites of cell-cell contact in stationary stage cells. MyoD is also present in filopodia, although the intensity is weaker than that of myoB staining. Despite persistence of myoD protein in the cells, myoD largely disappears from lamellipodia and cell-cell contact regions in aggregation stage cells, suggesting the occurrence of a developmentally regulated relocalization to the cytoplasm. While these results, along with the striking similarity in their tail domain structures, suggest that myoB and myoD have overlapping functions, differences in their localization in developing cells indicate that they have unique functions as well.