The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death.

The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt(-/-) cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57(low) tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57(low) cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57(low) cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anti-cancer immune response.

A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes.

We describe here a new variant of transposon Tn10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn10-LUK system has several important advantages. Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. Under favorable circumstances, usable lacZ protein fusions are created. Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.