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PMID: 9882627 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Subcellular localization of the Galphai3 protein and G alpha interacting protein, two proteins involved in the control of macroautophagy in human colon cancer HT-29 cells.

The Biochemical journal ·Vol. 337 ( Pt 2) ·1999-01-15 ·Pages 289-95

Petiot A, Ogier-Denis E, Bauvy C, Cluzeaud F, Vandewalle A, Codogno P

Abstract

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.

MeSH Terms
Autophagy Cell Compartmentation Cell Fractionation Colonic Neoplasms/metabolism Endoplasmic Reticulum/chemistry,ultrastructure Fluorescent Antibody Technique GTP-Binding Protein alpha Subunits, Gi-Go/genetics,isolation & purification Golgi Apparatus/chemistry,ultrastructure HT29 Cells Humans Phosphoproteins/genetics,immunology,isolation & purification RGS Proteins Recombinant Fusion Proteins/isolation & purification
Chemicals
Phosphoproteins RGS Proteins Recombinant Fusion Proteins regulator of G-protein signalling 19 GTP-Binding Protein alpha Subunits, Gi-Go
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Petiot A
INSERM U410, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, Paris 75018, France.
Ogier-Denis E
Bauvy C
Cluzeaud F
Vandewalle A
Codogno P
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
Published
1999-01-15
Pages
289-95
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1219964
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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