Intracellular expression of antibodies, intrabodies, in a cell is a powerful technology that has been used successfully to alter the function of the target antigen (Marasco, 1995). We have applied the intrabody technology to study the possible function and intracellular localisation of a highly conserved Saccharomyces cerevisiae Sem1 protein. The function of Sem1p is not known, but it interacts genetically with several components of the yeast secretory machinery (Jäntti et al., 1999). SEM1 is a single copy -gene and its deletion in 1278b makes this strain unable to grow on synthetic medium when the sole carbon source is galactose. Interestingly, the SEM1 human homologue DSS1, a possible candidate gene for the autosomal dominant form of split hand/split foot developmental disorder (Crackower et al., 1996), has been shown biochemically and by the two-hybrid assay to interact with the breast cancer susceptibility gene BRCA2 (Marston et al., 1999). In this work a Sem1p specific single chain antibody (scFv) isolated from a non-immunised human antibody phage display library was expressed intracellularily and targeted either to the cytosol or nucleus of yeast cells. Currently no conditional inactivating mutants of SEM1 gene exists. Thus inactivation of Sem1p function by intrabodies could mimic such mutants and could be used to study Sem1p function in yeast. Due to known difficulties in expression of functional intrabodies inside the cell we also studied the performance of different antibody formats as intrabodies. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the 1278b yeast strain in a similar manner as the deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localisation of the Fab intrabody with or without the nuclear localisation signal to the nucleus in Sem1p dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus Crackower, M. A., Scherer, S. W., Rommens, J. M., Hui, C. C., Poorkaj, P., Soder, S., Cobben, J. M., Hudgins, L., Evans, J. P. and Tsui, L. C. (1996). Hum Mol Genet 5, 571-9. Jäntti, J., Lahdenranta, J., Olkkonen, V. M., Söderlund, H. and Keränen, S. (1999). Proc Natl Acad Sci U S A 96, 909-14.Correction Proc Natl Acad Sci U S A 97, 14831 Marasco, W. A. (1995). Immunotechnology 1, 1-19. Marston, N. J., Richards, W. J., Hughes, D., Bertwistle, D., Marshall, C. J. and Ashworth, A. (1999). Mol Cell Biol 19, 4633-42.
|Number of pages||1|
|Publication status||Published - 2003|
|MoE publication type||Not Eligible|
|Event||EMBO Lecture Course New Developments in Genomics for Biomedicine - Brdo Estate, Kranj, Slovenia|
Duration: 8 Apr 2003 → 13 Apr 2003
|Course||EMBO Lecture Course New Developments in Genomics for Biomedicine|
|Period||8/04/03 → 13/04/03|