Yeast protein translocation complex

Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit

Jaana Toikkanen, Evelina Gatti, Kohji Takei, Markku Saloheimo, Vesa Olkkonen, Hans Söderlund, Pietro De Camilli, Sirkka Keränen

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion‐defective sec15–1 mutation. This gene encodes a protein homologous to the β subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane‐spanning region at the C‐terminus but no N‐terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61β subunit and 34% identity to the Arabidopsis thaliana Sec61β subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61–2 and sec61–3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N‐terminus exposed to the cytoplasm. The in vitro translated Seb1p was post‐translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature‐sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis. The sequences of SEB1 and SEB2 have been deposited in the EMBL database under Accession Numbers Z47789 and Z50012, respectively. The sequence of the Seb1 protein is identical to that of Sbh1p independently identified by Panzner et al. (Cell 81, 561–570. 1995) using a biochemical approach.
Original languageEnglish
Pages (from-to)425-438
Number of pages14
JournalYeast
Volume12
Issue number5
DOIs
Publication statusPublished - 1996
MoE publication typeA1 Journal article-refereed

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Fungal Proteins
Protein Transport
Genes
Proteins
Endoplasmic Reticulum
Membranes
Immunoelectron Microscopy
Protein Sorting Signals
Fluorescence Microscopy
Arabidopsis
Functional analysis
Cytoplasm
Plasmids
Complementary DNA
Clone Cells
Yeasts
Databases
Yeast
Phenotype
Amino Acids

Cite this

Toikkanen, Jaana ; Gatti, Evelina ; Takei, Kohji ; Saloheimo, Markku ; Olkkonen, Vesa ; Söderlund, Hans ; De Camilli, Pietro ; Keränen, Sirkka. / Yeast protein translocation complex : Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit. In: Yeast. 1996 ; Vol. 12, No. 5. pp. 425-438.
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title = "Yeast protein translocation complex: Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit",
abstract = "A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion‐defective sec15–1 mutation. This gene encodes a protein homologous to the β subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane‐spanning region at the C‐terminus but no N‐terminal signal sequence. Seb1p shows 30{\%} identity to the mammalian Sec61β subunit and 34{\%} identity to the Arabidopsis thaliana Sec61β subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61–2 and sec61–3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N‐terminus exposed to the cytoplasm. The in vitro translated Seb1p was post‐translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53{\%} identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature‐sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis. The sequences of SEB1 and SEB2 have been deposited in the EMBL database under Accession Numbers Z47789 and Z50012, respectively. The sequence of the Seb1 protein is identical to that of Sbh1p independently identified by Panzner et al. (Cell 81, 561–570. 1995) using a biochemical approach.",
author = "Jaana Toikkanen and Evelina Gatti and Kohji Takei and Markku Saloheimo and Vesa Olkkonen and Hans S{\"o}derlund and {De Camilli}, Pietro and Sirkka Ker{\"a}nen",
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Yeast protein translocation complex : Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit. / Toikkanen, Jaana; Gatti, Evelina; Takei, Kohji; Saloheimo, Markku; Olkkonen, Vesa; Söderlund, Hans; De Camilli, Pietro; Keränen, Sirkka.

In: Yeast, Vol. 12, No. 5, 1996, p. 425-438.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Yeast protein translocation complex

T2 - Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit

AU - Toikkanen, Jaana

AU - Gatti, Evelina

AU - Takei, Kohji

AU - Saloheimo, Markku

AU - Olkkonen, Vesa

AU - Söderlund, Hans

AU - De Camilli, Pietro

AU - Keränen, Sirkka

N1 - Project code: BEL4317

PY - 1996

Y1 - 1996

N2 - A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion‐defective sec15–1 mutation. This gene encodes a protein homologous to the β subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane‐spanning region at the C‐terminus but no N‐terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61β subunit and 34% identity to the Arabidopsis thaliana Sec61β subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61–2 and sec61–3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N‐terminus exposed to the cytoplasm. The in vitro translated Seb1p was post‐translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature‐sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis. The sequences of SEB1 and SEB2 have been deposited in the EMBL database under Accession Numbers Z47789 and Z50012, respectively. The sequence of the Seb1 protein is identical to that of Sbh1p independently identified by Panzner et al. (Cell 81, 561–570. 1995) using a biochemical approach.

AB - A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion‐defective sec15–1 mutation. This gene encodes a protein homologous to the β subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane‐spanning region at the C‐terminus but no N‐terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61β subunit and 34% identity to the Arabidopsis thaliana Sec61β subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61–2 and sec61–3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N‐terminus exposed to the cytoplasm. The in vitro translated Seb1p was post‐translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature‐sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis. The sequences of SEB1 and SEB2 have been deposited in the EMBL database under Accession Numbers Z47789 and Z50012, respectively. The sequence of the Seb1 protein is identical to that of Sbh1p independently identified by Panzner et al. (Cell 81, 561–570. 1995) using a biochemical approach.

U2 - 10.1002/(SICI)1097-0061(199604)12:5<425::AID-YEA924>3.0.CO;2-B

DO - 10.1002/(SICI)1097-0061(199604)12:5<425::AID-YEA924>3.0.CO;2-B

M3 - Article

VL - 12

SP - 425

EP - 438

JO - Yeast

JF - Yeast

SN - 0749-503X

IS - 5

ER -