Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

Jussi Jäntti, Sirkka Keränen, Jaana Toikkanen, Esa Kuismanen, Christian Ehnholm, Hans Söderlund, Vesa Olkkonen

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.
Original languageEnglish
Pages (from-to)3623-3633
Number of pages11
JournalJournal of Cell Science
Volume107
Issue number12
Publication statusPublished - 1994
MoE publication typeA1 Journal article-refereed

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Qa-SNARE Proteins
Intracellular Membranes
Yeasts
Membranes
Cell Membrane
Semliki forest virus
Brefeldin A
SNARE Proteins
Proteins
Membrane Fusion
Secretory Pathway
Biosynthetic Pathways
Golgi Apparatus
Cycloheximide
Protein Sorting Signals
Microsomes
Endoplasmic Reticulum
Fluorescent Antibody Technique
Membrane Proteins
Dogs

Cite this

Jäntti, J., Keränen, S., Toikkanen, J., Kuismanen, E., Ehnholm, C., Söderlund, H., & Olkkonen, V. (1994). Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells. Journal of Cell Science, 107(12), 3623-3633.
Jäntti, Jussi ; Keränen, Sirkka ; Toikkanen, Jaana ; Kuismanen, Esa ; Ehnholm, Christian ; Söderlund, Hans ; Olkkonen, Vesa. / Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells. In: Journal of Cell Science. 1994 ; Vol. 107, No. 12. pp. 3623-3633.
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abstract = "Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.",
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Jäntti, J, Keränen, S, Toikkanen, J, Kuismanen, E, Ehnholm, C, Söderlund, H & Olkkonen, V 1994, 'Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells', Journal of Cell Science, vol. 107, no. 12, pp. 3623-3633.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells. / Jäntti, Jussi; Keränen, Sirkka; Toikkanen, Jaana; Kuismanen, Esa; Ehnholm, Christian; Söderlund, Hans; Olkkonen, Vesa.

In: Journal of Cell Science, Vol. 107, No. 12, 1994, p. 3623-3633.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

AU - Jäntti, Jussi

AU - Keränen, Sirkka

AU - Toikkanen, Jaana

AU - Kuismanen, Esa

AU - Ehnholm, Christian

AU - Söderlund, Hans

AU - Olkkonen, Vesa

N1 - Project code: BEL4301

PY - 1994

Y1 - 1994

N2 - Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

AB - Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

M3 - Article

VL - 107

SP - 3623

EP - 3633

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 12

ER -

Jäntti J, Keränen S, Toikkanen J, Kuismanen E, Ehnholm C, Söderlund H et al. Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells. Journal of Cell Science. 1994;107(12):3623-3633.