Abstract. Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosorna brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.
The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected.
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