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. 1999 Jan 15;337(Pt 2):289–295.

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.

A Petiot 1, E Ogier-Denis 1, C Bauvy 1, F Cluzeaud 1, A Vandewalle 1, P Codogno 1
PMCID: PMC1219964  PMID: 9882627

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alb J. G., Jr, Kearns M. A., Bankaitis V. A. Phospholipid metabolism and membrane dynamics. Curr Opin Cell Biol. 1996 Aug;8(4):534–541. doi: 10.1016/s0955-0674(96)80032-9. [DOI] [PubMed] [Google Scholar]
  2. Berman D. M., Gilman A. G. Mammalian RGS proteins: barbarians at the gate. J Biol Chem. 1998 Jan 16;273(3):1269–1272. doi: 10.1074/jbc.273.3.1269. [DOI] [PubMed] [Google Scholar]
  3. Berman D. M., Wilkie T. M., Gilman A. G. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell. 1996 Aug 9;86(3):445–452. doi: 10.1016/s0092-8674(00)80117-8. [DOI] [PubMed] [Google Scholar]
  4. Blommaart E. F., Luiken J. J., Meijer A. J. Autophagic proteolysis: control and specificity. Histochem J. 1997 May;29(5):365–385. doi: 10.1023/a:1026486801018. [DOI] [PubMed] [Google Scholar]
  5. Bomsel M., Mostov K. Role of heterotrimeric G proteins in membrane traffic. Mol Biol Cell. 1992 Dec;3(12):1317–1328. doi: 10.1091/mbc.3.12.1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Codogno P., Ogier-Denis E., Houri J. J. Signal transduction pathways in macroautophagy. Cell Signal. 1997 Feb;9(2):125–130. doi: 10.1016/s0898-6568(96)00130-1. [DOI] [PubMed] [Google Scholar]
  7. De Camilli P., Emr S. D., McPherson P. S., Novick P. Phosphoinositides as regulators in membrane traffic. Science. 1996 Mar 15;271(5255):1533–1539. doi: 10.1126/science.271.5255.1533. [DOI] [PubMed] [Google Scholar]
  8. De Vries L., Elenko E., Hubler L., Jones T. L., Farquhar M. G. GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G alpha i subunits. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15203–15208. doi: 10.1073/pnas.93.26.15203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Vries L., Elenko E., McCaffery J. M., Fischer T., Hubler L., McQuistan T., Watson N., Farquhar M. G. RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles. Mol Biol Cell. 1998 May;9(5):1123–1134. doi: 10.1091/mbc.9.5.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Vries L., Mousli M., Wurmser A., Farquhar M. G. GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11916–11920. doi: 10.1073/pnas.92.25.11916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunn W. A., Jr Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol. 1994 Apr;4(4):139–143. doi: 10.1016/0962-8924(94)90069-8. [DOI] [PubMed] [Google Scholar]
  12. Dunn W. A., Jr Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1923–1933. doi: 10.1083/jcb.110.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunn W. A., Jr Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1935–1945. doi: 10.1083/jcb.110.6.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fengsrud M., Roos N., Berg T., Liou W., Slot J. W., Seglen P. O. Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of vinblastine and asparagine on vacuole distributions. Exp Cell Res. 1995 Dec;221(2):504–519. doi: 10.1006/excr.1995.1402. [DOI] [PubMed] [Google Scholar]
  15. Helms J. B. Role of heterotrimeric GTP binding proteins in vesicular protein transport: indications for both classical and alternative G protein cycles. FEBS Lett. 1995 Aug 1;369(1):84–88. doi: 10.1016/0014-5793(95)00620-o. [DOI] [PubMed] [Google Scholar]
  16. Houri J. J., Ogier-Denis E., De Stefanis D., Bauvy C., Baccino F. M., Isidoro C., Codogno P. Differentiation-dependent autophagy controls the fate of newly synthesized N-linked glycoproteins in the colon adenocarcinoma HT-29 cell line. Biochem J. 1995 Jul 15;309(Pt 2):521–527. doi: 10.1042/bj3090521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang C., Hepler J. R., Gilman A. G., Mumby S. M. Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6159–6163. doi: 10.1073/pnas.94.12.6159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jamora C., Takizawa P. A., Zaarour R. F., Denesvre C., Faulkner D. J., Malhotra V. Regulation of Golgi structure through heterotrimeric G proteins. Cell. 1997 Nov 28;91(5):617–626. doi: 10.1016/s0092-8674(00)80449-3. [DOI] [PubMed] [Google Scholar]
  19. Kadowaki M., Venerando R., Miotto G., Mortimore G. E. De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs. J Biol Chem. 1994 Feb 4;269(5):3703–3710. [PubMed] [Google Scholar]
  20. Koelle M. R. A new family of G-protein regulators - the RGS proteins. Curr Opin Cell Biol. 1997 Apr;9(2):143–147. doi: 10.1016/s0955-0674(97)80055-5. [DOI] [PubMed] [Google Scholar]
  21. Lawrence B. P., Brown W. J. Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes. J Cell Sci. 1992 Jul;102(Pt 3):515–526. doi: 10.1242/jcs.102.3.515. [DOI] [PubMed] [Google Scholar]
  22. Liou W., Geuze H. J., Geelen M. J., Slot J. W. The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. J Cell Biol. 1997 Jan 13;136(1):61–70. doi: 10.1083/jcb.136.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liscovitch M., Cantley L. C. Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell. 1995 Jun 2;81(5):659–662. doi: 10.1016/0092-8674(95)90525-1. [DOI] [PubMed] [Google Scholar]
  24. Lodish H. F., Kong N., Hirani S., Rasmussen J. A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex. J Cell Biol. 1987 Feb;104(2):221–230. doi: 10.1083/jcb.104.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lucocq J., Walker D. Evidence for fusion between multilamellar endosomes and autophagosomes in HeLa cells. Eur J Cell Biol. 1997 Apr;72(4):307–313. [PubMed] [Google Scholar]
  26. Montmayeur J. P., Borrelli E. Targeting of G alpha i2 to the Golgi by alternative spliced carboxyl-terminal region. Science. 1994 Jan 7;263(5143):95–98. doi: 10.1126/science.8272874. [DOI] [PubMed] [Google Scholar]
  27. Moss J., Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol Relat Areas Mol Biol. 1988;61:303–379. doi: 10.1002/9780470123072.ch6. [DOI] [PubMed] [Google Scholar]
  28. Ogier-Denis E., Bauvy C., Houri J. J., Codogno P. Evidence for a dual control of macroautophagic sequestration and intracellular trafficking of N-linked glycoproteins by the trimeric G(i3) protein in HT-29 cells. Biochem Biophys Res Commun. 1997 Jun 9;235(1):166–170. doi: 10.1006/bbrc.1997.6727. [DOI] [PubMed] [Google Scholar]
  29. Ogier-Denis E., Couvineau A., Maoret J. J., Houri J. J., Bauvy C., De Stefanis D., Isidoro C., Laburthe M., Codogno P. A heterotrimeric Gi3-protein controls autophagic sequestration in the human colon cancer cell line HT-29. J Biol Chem. 1995 Jan 6;270(1):13–16. doi: 10.1074/jbc.270.1.13. [DOI] [PubMed] [Google Scholar]
  30. Ogier-Denis E., Houri J. J., Bauvy C., Codogno P. Guanine nucleotide exchange on heterotrimeric Gi3 protein controls autophagic sequestration in HT-29 cells. J Biol Chem. 1996 Nov 8;271(45):28593–28600. doi: 10.1074/jbc.271.45.28593. [DOI] [PubMed] [Google Scholar]
  31. Ogier-Denis E., Petiot A., Bauvy C., Codogno P. Control of the expression and activity of the Galpha-interacting protein (GAIP) in human intestinal cells. J Biol Chem. 1997 Sep 26;272(39):24599–24603. doi: 10.1074/jbc.272.39.24599. [DOI] [PubMed] [Google Scholar]
  32. Punnonen E. L., Autio S., Kaija H., Reunanen H. Autophagic vacuoles fuse with the prelysosomal compartment in cultured rat fibroblasts. Eur J Cell Biol. 1993 Jun;61(1):54–66. [PubMed] [Google Scholar]
  33. Purhonen P., Pursiainen K., Reunanen H. Effects of brefeldin A on autophagy in cultured rat fibroblasts. Eur J Cell Biol. 1997 Sep;74(1):63–67. [PubMed] [Google Scholar]
  34. Rabouille C., Strous G. J., Crapo J. D., Geuze H. J., Slot J. W. The differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry. J Cell Biol. 1993 Feb;120(4):897–908. doi: 10.1083/jcb.120.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seglen P. O., Bohley P. Autophagy and other vacuolar protein degradation mechanisms. Experientia. 1992 Feb 15;48(2):158–172. doi: 10.1007/BF01923509. [DOI] [PubMed] [Google Scholar]
  36. Seglen P. O., Gordon P. B. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1889–1892. doi: 10.1073/pnas.79.6.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stephens L., Smrcka A., Cooke F. T., Jackson T. R., Sternweis P. C., Hawkins P. T. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 1994 Apr 8;77(1):83–93. doi: 10.1016/0092-8674(94)90237-2. [DOI] [PubMed] [Google Scholar]
  38. Stow J. L., de Almeida J. B., Narula N., Holtzman E. J., Ercolani L., Ausiello D. A. A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells. J Cell Biol. 1991 Sep;114(6):1113–1124. doi: 10.1083/jcb.114.6.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stoyanov B., Volinia S., Hanck T., Rubio I., Loubtchenkov M., Malek D., Stoyanova S., Vanhaesebroeck B., Dhand R., Nürnberg B. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 1995 Aug 4;269(5224):690–693. doi: 10.1126/science.7624799. [DOI] [PubMed] [Google Scholar]
  40. Strømhaug P. E., Seglen P. O. Evidence for acidity of prelysosomal autophagic/endocytic vacuoles (amphisomes). Biochem J. 1993 Apr 1;291(Pt 1):115–121. doi: 10.1042/bj2910115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tooze J., Hollinshead M., Ludwig T., Howell K., Hoflack B., Kern H. In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome. J Cell Biol. 1990 Aug;111(2):329–345. doi: 10.1083/jcb.111.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weibel E. R., Stäubli W., Gnägi H. R., Hess F. A. Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver. J Cell Biol. 1969 Jul;42(1):68–91. doi: 10.1083/jcb.42.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wendland B., Scheller R. H. Secretion in AtT-20 cells stably transfected with soluble synaptotagmins. Mol Endocrinol. 1994 Aug;8(8):1070–1082. doi: 10.1210/mend.8.8.7997233. [DOI] [PubMed] [Google Scholar]
  44. Yamaguchi T., Yamamoto A., Furuno A., Hatsuzawa K., Tani K., Himeno M., Tagaya M. Possible involvement of heterotrimeric G proteins in the organization of the Golgi apparatus. J Biol Chem. 1997 Oct 3;272(40):25260–25266. doi: 10.1074/jbc.272.40.25260. [DOI] [PubMed] [Google Scholar]
  45. Yamamoto A., Masaki R., Fukui Y., Tashiro Y. Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes. J Histochem Cytochem. 1990 Nov;38(11):1571–1581. doi: 10.1177/38.11.2212617. [DOI] [PubMed] [Google Scholar]
  46. Yamamoto A., Masaki R., Tashiro Y. Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry. J Histochem Cytochem. 1990 Apr;38(4):573–580. doi: 10.1177/38.4.2319125. [DOI] [PubMed] [Google Scholar]
  47. Zweibaum A., Pinto M., Chevalier G., Dussaulx E., Triadou N., Lacroix B., Haffen K., Brun J. L., Rousset M. Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose. J Cell Physiol. 1985 Jan;122(1):21–29. doi: 10.1002/jcp.1041220105. [DOI] [PubMed] [Google Scholar]
  48. de Almeida J. B., Holtzman E. J., Peters P., Ercolani L., Ausiello D. A., Stow J. L. Targeting of chimeric G alpha i proteins to specific membrane domains. J Cell Sci. 1994 Mar;107(Pt 3):507–515. doi: 10.1242/jcs.107.3.507. [DOI] [PubMed] [Google Scholar]

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