IMPORT OF A DHFR HYBRID PROTEIN INTO GLYCOSOMES IN-VIVO IS NOT INHIBITED BY THE FOLATE-ANALOG AMINOPTERIN

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membran es. We have studied the import of dihydrofolate reductase fusion prote ins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosom al phosphoglycerate kinase for glycosomes, and the cleavable presequen ces of yeast cytochrome b(2) or cytochrome oxidase subunit IV for mito chondria. Upon addition of aminopterin, a folate analogue that stabili zes the dihydrofolate reductase moiety, import of the fusion protein t argeted to glycosomes was not inhibited, although the results of prote ase protection assays showed that the fusion protein could bind the dr ug. Under the same conditions, import of a DHFR fusion protein targete d to mitochondria was inhibited by aminopterin. When DHFR fusion prote ins targeted simultaneously to both glycosomes and mitochondria were e xpressed, import into mitochondria was inhibited by aminopterin, where as uptake of the same proteins into glycosomes was either unaffected o r slightly increased. These findings suggest that the glycosomes posse ss either a strong unfolding activity or an unusually large or flexibl e translocation channel.