Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Nov 15;280(Pt 1):171–178. doi: 10.1042/bj2800171

Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes.

L M Traub 1, E Shai 1, R Sagi-Eisenberg 1
PMCID: PMC1130616  PMID: 1741744

Abstract

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.

Full text

PDF
171

Images in this article

173Fig. 1.
on p.173
173Fig. 2.
on p.173
174Fig. 3.
on p.174
174Fig. 4.
on p.174
175Fig. 5.
on p.175
175Fig. 6.
on p.175
176Fig. 7.
on p.176

Selected References

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

  1. Allan V. J., Kreis T. E. A microtubule-binding protein associated with membranes of the Golgi apparatus. J Cell Biol. 1986 Dec;103(6 Pt 1):2229–2239. doi: 10.1083/jcb.103.6.2229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aridor M., Sagi-Eisenberg R. Neomycin is a potent secretagogue of mast cells that directly activates a GTP-binding protein involved in exocytosis. J Cell Biol. 1990 Dec;111(6 Pt 2):2885–2891. doi: 10.1083/jcb.111.6.2885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aridor M., Traub L. M., Sagi-Eisenberg R. Exocytosis in mast cells by basic secretagogues: evidence for direct activation of GTP-binding proteins. J Cell Biol. 1990 Sep;111(3):909–917. doi: 10.1083/jcb.111.3.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beck K. A., Keen J. H. Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem. 1991 Mar 5;266(7):4442–4447. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Cerione R. A., Kroll S., Rajaram R., Unson C., Goldsmith P., Spiegel A. M. An antibody directed against the carboxyl-terminal decapeptide of the alpha subunit of the retinal GTP-binding protein, transducin. Effects on transducin function. J Biol Chem. 1988 Jul 5;263(19):9345–9352. [PubMed] [Google Scholar]
  7. Cheung A. H., Sigal I. S., Dixon R. A., Strader C. D. Agonist-promoted sequestration of the beta 2-adrenergic receptor requires regions involved in functional coupling with Gs. Mol Pharmacol. 1989 Jan;35(1):132–138. [PubMed] [Google Scholar]
  8. Donaldson J. G., Lippincott-Schwartz J., Bloom G. S., Kreis T. E., Klausner R. D. Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol. 1990 Dec;111(6 Pt 1):2295–2306. doi: 10.1083/jcb.111.6.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donaldson J. G., Lippincott-Schwartz J., Klausner R. D. Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus. J Cell Biol. 1991 Feb;112(4):579–588. doi: 10.1083/jcb.112.4.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duden R., Griffiths G., Frank R., Argos P., Kreis T. E. Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell. 1991 Feb 8;64(3):649–665. doi: 10.1016/0092-8674(91)90248-w. [DOI] [PubMed] [Google Scholar]
  11. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gershoni J. M. Protein blotting: a manual. Methods Biochem Anal. 1988;33:1–58. doi: 10.1002/9780470110546.ch1. [DOI] [PubMed] [Google Scholar]
  13. Goldsmith P., Gierschik P., Milligan G., Unson C. G., Vinitsky R., Malech H. L., Spiegel A. M. Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain. J Biol Chem. 1987 Oct 25;262(30):14683–14688. [PubMed] [Google Scholar]
  14. Hamm H. E., Deretic D., Mazzoni M. R., Moore C. A., Takahashi J. S., Rasenick M. M. A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase. J Biol Chem. 1989 Jul 5;264(19):11475–11482. [PubMed] [Google Scholar]
  15. Higashijima T., Burnier J., Ross E. M. Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. J Biol Chem. 1990 Aug 25;265(24):14176–14186. [PubMed] [Google Scholar]
  16. Hortsch M., Meyer D. I. Immunochemical analysis of rough and smooth microsomes from rat liver. Segregation of docking protein in rough membranes. Eur J Biochem. 1985 Aug 1;150(3):559–564. doi: 10.1111/j.1432-1033.1985.tb09057.x. [DOI] [PubMed] [Google Scholar]
  17. Keen J. H. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem. 1990;59:415–438. doi: 10.1146/annurev.bi.59.070190.002215. [DOI] [PubMed] [Google Scholar]
  18. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Linderman J. J., Lauffenburger D. A. Analysis of intracellular receptor/ligand sorting in endosomes. J Theor Biol. 1988 May 21;132(2):203–245. doi: 10.1016/s0022-5193(88)80157-7. [DOI] [PubMed] [Google Scholar]
  21. Masters S. B., Stroud R. M., Bourne H. R. Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains. Protein Eng. 1986 Oct-Nov;1(1):47–54. [PubMed] [Google Scholar]
  22. McKenzie F. R., Kelly E. C., Unson C. G., Spiegel A. M., Milligan G. Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. Biochem J. 1988 Feb 1;249(3):653–659. doi: 10.1042/bj2490653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morris S. A., Ahle S., Ungewickell E. Clathrin-coated vesicles. Curr Opin Cell Biol. 1989 Aug;1(4):684–690. doi: 10.1016/0955-0674(89)90034-3. [DOI] [PubMed] [Google Scholar]
  24. Nilsson L., Nygård O. Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2. Eur J Biochem. 1985 Apr 15;148(2):299–304. doi: 10.1111/j.1432-1033.1985.tb08839.x. [DOI] [PubMed] [Google Scholar]
  25. Palm D., Münch G., Malek D., Dees C., Hekman M. Identification of a Gs-protein coupling domain to the beta-adrenoceptor using site-specific synthetic peptides. Carboxyl terminus of Gs alpha is involved in coupling to beta-adrenoceptors. FEBS Lett. 1990 Feb 26;261(2):294–298. doi: 10.1016/0014-5793(90)80575-4. [DOI] [PubMed] [Google Scholar]
  26. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  27. Ryazanov A. G., Shestakova E. A., Natapov P. G. Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation. Nature. 1988 Jul 14;334(6178):170–173. doi: 10.1038/334170a0. [DOI] [PubMed] [Google Scholar]
  28. Savitz A. J., Meyer D. I. Identification of a ribosome receptor in the rough endoplasmic reticulum. Nature. 1990 Aug 9;346(6284):540–544. doi: 10.1038/346540a0. [DOI] [PubMed] [Google Scholar]
  29. Shepherd V. L. Intracellular pathways and mechanisms of sorting in receptor-mediated endocytosis. Trends Pharmacol Sci. 1989 Nov;10(11):458–462. doi: 10.1016/S0165-6147(89)80011-2. [DOI] [PubMed] [Google Scholar]
  30. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Simonds W. F., Goldsmith P. K., Woodard C. J., Unson C. G., Spiegel A. M. Receptor and effector interactions of Gs. Functional studies with antibodies to the alpha s carboxyl-terminal decapeptide. FEBS Lett. 1989 Jun 5;249(2):189–194. doi: 10.1016/0014-5793(89)80622-2. [DOI] [PubMed] [Google Scholar]
  32. Traub L. M., Evans W. H., Sagi-Eisenberg R. A novel 100 kDa protein, localized to receptor-enriched endosomes, is immunologically related to the signal-transducing guanine-nucleotide-binding proteins Gt and Gi. Biochem J. 1990 Dec 1;272(2):453–458. doi: 10.1042/bj2720453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Udrisar D., Rodbell M. Microsomal and cytosolic fractions of guinea pig hepatocytes contain 100-kilodalton GTP-binding proteins reactive with antisera against alpha subunits of stimulatory and inhibitory heterotrimeric GTP-binding proteins. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6321–6325. doi: 10.1073/pnas.87.16.6321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Valiquette M., Bonin H., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5089–5093. doi: 10.1073/pnas.87.13.5089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Waters M. G., Serafini T., Rothman J. E. 'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature. 1991 Jan 17;349(6306):248–251. doi: 10.1038/349248a0. [DOI] [PubMed] [Google Scholar]
  36. Wilden U., Hall S. W., Kühn H. Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1174–1178. doi: 10.1073/pnas.83.5.1174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wistow G. J., Katial A., Craft C., Shinohara T. Sequence analysis of bovine retinal S-antigen. Relationships with alpha-transducin and G-proteins. FEBS Lett. 1986 Feb 3;196(1):23–28. doi: 10.1016/0014-5793(86)80207-1. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES