Functional inactivation of the conserved Sem1p in yeast by intrabodies.
Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. 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 sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization 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.
Reinman
Mirka
M
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland. mirka.reinman@vtt.fi
Jäntti
Jussi
J
Alfthan
Kaija
K
Keränen
Sirkka
S
Söderlund
Hans
H
Takkinen
Kristiina
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; C117424:SEM1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex