Functional inactivation of the conserved Sem1p in yeast by intrabodies

Mirka Reinman (Corresponding Author), Jussi Jäntti, Kaija Alfthan, Sirkka Keränen, Hans Söderlund, Kristiina Takkinen

Research output: Contribution to journalArticleScientificpeer-review

12 Citations (Scopus)

Abstract

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 Σ1278b 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.
Original languageEnglish
Pages (from-to)1071-1084
Number of pages14
JournalYeast
Volume20
Issue number12
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

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Yeast
Yeasts
Cells
Antibodies
Saccharomyces cerevisiae Proteins
Nuclear Localization Signals
Immunoglobulin Fragments
Bacteriophages
Cell Nucleus Active Transport
Gene Deletion
Cell Nucleus
Cytosol
Libraries
Tumors
Genes
Display devices
Technology
Proteins
Growth
Neoplasms

Keywords

  • Fab fragment
  • intracellular antibody
  • Sem1p
  • DSS1
  • nuclear localization

Cite this

Reinman, Mirka ; Jäntti, Jussi ; Alfthan, Kaija ; Keränen, Sirkka ; Söderlund, Hans ; Takkinen, Kristiina. / Functional inactivation of the conserved Sem1p in yeast by intrabodies. In: Yeast. 2003 ; Vol. 20, No. 12. pp. 1071-1084.
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abstract = "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 Σ1278b 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.",
keywords = "Fab fragment, intracellular antibody, Sem1p, DSS1, nuclear localization",
author = "Mirka Reinman and Jussi J{\"a}ntti and Kaija Alfthan and Sirkka Ker{\"a}nen and Hans S{\"o}derlund and Kristiina Takkinen",
year = "2003",
doi = "10.1002/yea.1027",
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Reinman, M, Jäntti, J, Alfthan, K, Keränen, S, Söderlund, H & Takkinen, K 2003, 'Functional inactivation of the conserved Sem1p in yeast by intrabodies', Yeast, vol. 20, no. 12, pp. 1071-1084. https://doi.org/10.1002/yea.1027

Functional inactivation of the conserved Sem1p in yeast by intrabodies. / Reinman, Mirka (Corresponding Author); Jäntti, Jussi; Alfthan, Kaija; Keränen, Sirkka; Söderlund, Hans; Takkinen, Kristiina.

In: Yeast, Vol. 20, No. 12, 2003, p. 1071-1084.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Functional inactivation of the conserved Sem1p in yeast by intrabodies

AU - Reinman, Mirka

AU - Jäntti, Jussi

AU - Alfthan, Kaija

AU - Keränen, Sirkka

AU - Söderlund, Hans

AU - Takkinen, Kristiina

PY - 2003

Y1 - 2003

N2 - 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 Σ1278b 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.

AB - 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 Σ1278b 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.

KW - Fab fragment

KW - intracellular antibody

KW - Sem1p

KW - DSS1

KW - nuclear localization

U2 - 10.1002/yea.1027

DO - 10.1002/yea.1027

M3 - Article

VL - 20

SP - 1071

EP - 1084

JO - Yeast

JF - Yeast

SN - 0749-503X

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ER -