Targeting of chimeric Gαi proteins to specific membrane domains

Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous Gαi-2 is localized on the basolateral plasma membrane, whereas Gαi-3...

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Principais autores: Almeida, Jose Bruno de, Holtzman, Eliezer J., Peters, Philip, Ercolani, Louis, Ausiello, Dennis A., Stow, Jennifer L.
Formato: article
Idioma:English
Publicado em: Journal of Cell Science
Assuntos:
targeting signal
g protein
plasma membrane
immunofluorescence
golgi
Endereço do item:https://repositorio.ufrn.br/handle/123456789/54378
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spelling ri-123456789-543782023-08-03T19:39:05Z Targeting of chimeric Gαi proteins to specific membrane domains Almeida, Jose Bruno de Holtzman, Eliezer J. Peters, Philip Ercolani, Louis Ausiello, Dennis A. Stow, Jennifer L. targeting signal g protein plasma membrane immunofluorescence golgi Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous Gαi-2 is localized on the basolateral plasma membrane, whereas Gαi-3 is localized on Golgi membranes. The targeting of these highly homologous Gαi proteins to distinct membrane domains was studied by the transfection and expression of chimeric Gαi proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for Gαi-3 and Gαi-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothioneinI promotor. Stably transfected cell lines were produced that expressed either Gαi-2/3 or Gαi-3/2 chimeric proteins. Chimeric and endogenous Gαi proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric Gαi proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous Gαi proteins. Using confocal microscopy and image analysis we localized Gαi-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric Gαi-3/2 protein was then shown to colocalize with endognenous Gαi-2 in the same lateral plasma membrane domain. The chimeric Gαi-2/3 protein colocalized with endogenous Gαi-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric Gαi proteins were targeted to the same membrane domains as endogenous Gαi proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of Gαi proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins 2023-08-03T19:39:04Z 2023-08-03T19:39:04Z 1994 article ALMEIDA, Jose Bruno de; Eliezer J. Holtzman ; AUSIELLO, D. A. ; STOW, J. L. . Targeting of chimeric Gai proteins to specific membrane domains. Journal of Cell Science, v. 107, p. 507-515, 1994. Disponível em: https://espace.library.uq.edu.au/view/UQ:311108 Acesso em: 31 jul 2023 0021-9533 https://repositorio.ufrn.br/handle/123456789/54378 en application/pdf Journal of Cell Science
institution Repositório Institucional
collection RI - UFRN
language English
topic targeting signal
g protein
plasma membrane
immunofluorescence
golgi
spellingShingle targeting signal
g protein
plasma membrane
immunofluorescence
golgi
Almeida, Jose Bruno de
Holtzman, Eliezer J.
Peters, Philip
Ercolani, Louis
Ausiello, Dennis A.
Stow, Jennifer L.
Targeting of chimeric Gαi proteins to specific membrane domains
description Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous Gαi-2 is localized on the basolateral plasma membrane, whereas Gαi-3 is localized on Golgi membranes. The targeting of these highly homologous Gαi proteins to distinct membrane domains was studied by the transfection and expression of chimeric Gαi proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for Gαi-3 and Gαi-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothioneinI promotor. Stably transfected cell lines were produced that expressed either Gαi-2/3 or Gαi-3/2 chimeric proteins. Chimeric and endogenous Gαi proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric Gαi proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous Gαi proteins. Using confocal microscopy and image analysis we localized Gαi-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric Gαi-3/2 protein was then shown to colocalize with endognenous Gαi-2 in the same lateral plasma membrane domain. The chimeric Gαi-2/3 protein colocalized with endogenous Gαi-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric Gαi proteins were targeted to the same membrane domains as endogenous Gαi proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of Gαi proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins
format article
author Almeida, Jose Bruno de
Holtzman, Eliezer J.
Peters, Philip
Ercolani, Louis
Ausiello, Dennis A.
Stow, Jennifer L.
author_facet Almeida, Jose Bruno de
Holtzman, Eliezer J.
Peters, Philip
Ercolani, Louis
Ausiello, Dennis A.
Stow, Jennifer L.
author_sort Almeida, Jose Bruno de
title Targeting of chimeric Gαi proteins to specific membrane domains
title_short Targeting of chimeric Gαi proteins to specific membrane domains
title_full Targeting of chimeric Gαi proteins to specific membrane domains
title_fullStr Targeting of chimeric Gαi proteins to specific membrane domains
title_full_unstemmed Targeting of chimeric Gαi proteins to specific membrane domains
title_sort targeting of chimeric gαi proteins to specific membrane domains
publisher Journal of Cell Science
publishDate 2023
url https://repositorio.ufrn.br/handle/123456789/54378
work_keys_str_mv AT almeidajosebrunode targetingofchimericgaiproteinstospecificmembranedomains
AT holtzmaneliezerj targetingofchimericgaiproteinstospecificmembranedomains
AT petersphilip targetingofchimericgaiproteinstospecificmembranedomains
AT ercolanilouis targetingofchimericgaiproteinstospecificmembranedomains
AT ausiellodennisa targetingofchimericgaiproteinstospecificmembranedomains
AT stowjenniferl targetingofchimericgaiproteinstospecificmembranedomains
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