The cytochrome b5 tail anchors and stabilizes subdomains of human DNA topoisomerase II alpha in the cytoplasm of retrovirally infected mammalian cells.
DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIalpha are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIalpha and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b5 anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1-266) is weakly and diffusely expressed, addition of the cytb5 anchor (1-266-b5) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1-426). A C-terminal domain (aa 1030-1504) of human topo IIalpha containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb5 anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.
Soltermann
A
A
Swiss Institute for Experimental Cancer Research, Epalinges/Lausanne, CH-1066, Switzerland.
Ernst
A
A
Leroy
D
D
Stahel
R A
RA
Gasser
S M
SM
D000818:Animals; D000951:Antigens, Neoplasm; D002460:Cell Line; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism / Q000821:virology*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005047:Etoposide / Q000494:pharmacology; D018014:Gene Transfer Techniques; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019913:Nuclear Localization Signals / Q000235:genetics; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000187:drug effects / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D019913:Nuclear Localization Signals; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005047:Etoposide; D015786:Cytochromes b5; C085706:FLAG peptide; D004250:DNA Topoisomerases, Type II