Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin.
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 Trypanosoma 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.
Häusler
T
T
Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
Stierhof
Y D
YD
Wirtz
E
E
Clayton
C
C
D000630:Aminopterin / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome); D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D015388:Organelles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D055503:Protein Multimerization; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000648:ultrastructure
D017931:DNA Primers; D005493:Folic Acid Antagonists; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase; D010735:Phosphoglycerate Kinase; D000630:Aminopterin