Key points are not available for this paper at this time.
Coat proteins of the COP family were recently shown by us and others to be involved in membrane transport in the endocytic pathway, in addition to their known functions in the biosynthetic pathway. We have also shown that membrane association of endosomal COPs depends on the acidic endosomal pH, in contrast to biosynthetic COPs. In this paper, we report that both membrane recruitment of endosomal COPs and in vitro biogenesis of transport intermediates destined for late endosomes, depend on a cytosolic factor, which we identified as the small GTP-binding protein ARF1. Our data indicate that ARF1 does not act via activation of an endosomal phospholipase D. We also find that ARF1 membrane association is regulated by the endosomal pH, and that this controls the pH-dependent association of endosomal COPs. These studies thus show that ARF1 regulates COP functions in the endocytic pathway, and indicate that ARF1 acts as the cytosolic component of a transmembrane pH-sensing mechanism. Coat proteins of the COP family were recently shown by us and others to be involved in membrane transport in the endocytic pathway, in addition to their known functions in the biosynthetic pathway. We have also shown that membrane association of endosomal COPs depends on the acidic endosomal pH, in contrast to biosynthetic COPs. In this paper, we report that both membrane recruitment of endosomal COPs and in vitro biogenesis of transport intermediates destined for late endosomes, depend on a cytosolic factor, which we identified as the small GTP-binding protein ARF1. Our data indicate that ARF1 does not act via activation of an endosomal phospholipase D. We also find that ARF1 membrane association is regulated by the endosomal pH, and that this controls the pH-dependent association of endosomal COPs. These studies thus show that ARF1 regulates COP functions in the endocytic pathway, and indicate that ARF1 acts as the cytosolic component of a transmembrane pH-sensing mechanism. endosomal carrier vesicles/multivesicular body ADP-ribosylation factor phospholipase D phosphatidic acid phosphatidylcholine baby hamster kidney horseradish peroxidase guanosine 5′-3-O-(thio)- triphosphate homogenization buffer In animal cells, the dynamic flow of proteins and lipids between the plasma membrane and early endosome is maintained by rapid internalization and recycling processes. Most internalized cell surface molecules are recycled, whereas molecules which are destined to be degraded, including all down-regulated cell surface receptors, are sorted within early endosomes, and then transported toward late endosomes and lysosomes (1.Gruenberg J. Maxfield F. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (550) Google Scholar, 2.Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-626Crossref PubMed Scopus (1331) Google Scholar). Transport from early to late endosomes is mediated by relatively large carrier vesicles (0.4–0.5 μm diameter) with a typical multivesicular appearance (3.Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (453) Google Scholar), which will be referred to here as endosomal carrier vesicles/multivesicular bodies (ECV/MVBs).1 Once formed on early endosomal membranes, ECV/MVBs move toward late endosome, and this movement depends on intact microtubules and motor proteins (3.Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (453) Google Scholar, 4.Aniento F. Emans N. Griffiths G. Gruenberg J. J. Cell Biol. 1993; 123: 1373-1388Crossref PubMed Scopus (366) Google Scholar, 5.Bomsel M. Parton R. Kuznetsov S.A. Schroer T.A. Gruenberg J. Cell. 1990; 62: 719-731Abstract Full Text PDF PubMed Scopus (226) Google Scholar). Eventually, ECVs dock onto and fuse with late endosomes, in a process which depends on α soluble N-ethylmaleimide sensitive factor (NSF) attachment protein, NSF, and perhaps another member of the triple A ATPase family (6.Robinson L. Aniento F. Gruenberg J. J. Cell Sci. 1997; 110: 2079-2087Crossref PubMed Google Scholar), as well as presumably the small GTPase rab7 (7.Feng Y. Press B. Wandinger-Ness A. J. Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (529) Google Scholar).In vivo and in vitro studies have shown that ECV/MVB formation on early endosomes depends on some, but not all, components of the COP-I coat complex (8.Aniento F. Gu F. Parton R. Gruenberg J. J. Cell Biol. 1996; 133: 29-41Crossref PubMed Scopus (314) Google Scholar, 9.Gu F. Aniento F. Parton R. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (138) Google Scholar, 10.Whitney J.A. Gomez M. Sheff D. Kreis T.E. Mellman I. Cell. 1995; 83: 703-713Abstract Full Text PDF PubMed Scopus (265) Google Scholar), which is known to be also involved in the early secretory pathway (11.Lowe M. Kreis T.E. Biochim. Biophys. Acta. 1998; 1404: 53-66Crossref PubMed Scopus (81) Google Scholar). Recent studies, in fact, indicated that endosomal COPs contribute to the down-regulation of the Nef-CD4 complex, via direct interactions between Nef and βCOP (12.Piguet V. Gu F. Foti M. Demaurex N. Gruenberg J. Carpentier J.-L. Trono D. Cell. 1999; 97: 63-73Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Similarly, the AP3 adaptor complex also appears to be involved in more than one pathway, namely transport toward late endosomes/lysosomes (13.Le Borgne R. Hoflack B. Curr. Opin. Cell Biol. 1998; 10: 499-503Crossref PubMed Scopus (91) Google Scholar) and synaptic vesicle formation (14.Faundez V. Horng J.T. Kelly R.B. Cell. 1998; 93: 423-432Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In addition to endosomal COPs, ECV/MVB formation also depends on the acidification properties of early endosomes (15.Clague M. Urbé S. Aniento F. Gruenberg J. J. Biol. Chem. 1994; 269: 21-24Abstract Full Text PDF PubMed Google Scholar). These pH- and COP-dependent processes are related functionally and biochemically, since COP association to endosomal membranes, but not to biosynthetic membranes, is itself pH-sensitive (8.Aniento F. Gu F. Parton R. Gruenberg J. J. Cell Biol. 1996; 133: 29-41Crossref PubMed Scopus (314) Google Scholar, 9.Gu F. Aniento F. Parton R. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (138) Google Scholar). These observations lead us to propose that a transmembrane pH-sensor regulates COP association to endosomes, thereby signaling the onset of the degradation pathway on early endosomal membranes.In the present paper, we have further dissected the molecular process which regulates membrane association of endosomal COPs. Our data show that the small GTP-binding protein ARF1 is required for COP recruitment onto endosomes, and for ECV/MVB biogenesis from donor early endosomal membranes in vitro. We find that PLD and phosphatidic acid are not involved in this process, indicating that ARF1 does not act via PLD on early endosomal membranes, in contrast to biosynthetic COPs (16.Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (328) Google Scholar). Moreover, our data show that ARF1 recruitment onto endosomes depends on the acidic lumenal pH, in agreement with previous studies on ARF binding to microsomes (17.Zeuzem S. Feick P. Zimmermann P. Haase W. Kahn R.A. Schulz I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6619-6623Crossref PubMed Scopus (76) Google Scholar), and that this mechanism accounts for the pH dependence of endosomal COPs association to membranes. Our data thus show that ARF1 mediates COP binding to endosomes and ECV/MVB biogenesis, in a pH-dependent, but PLD-independent, process. ARF1 thus appears to act as the cytosolic component relaying lumenal pH variations to endosomal COPs during ECV/MVB biogenesis. In animal cells, the dynamic flow of proteins and lipids between the plasma membrane and early endosome is maintained by rapid internalization and recycling processes. Most internalized cell surface molecules are recycled, whereas molecules which are destined to be degraded, including all down-regulated cell surface receptors, are sorted within early endosomes, and then transported toward late endosomes and lysosomes (1.Gruenberg J. Maxfield F. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (550) Google Scholar, 2.Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-626Crossref PubMed Scopus (1331) Google Scholar). Transport from early to late endosomes is mediated by relatively large carrier vesicles (0.4–0.5 μm diameter) with a typical multivesicular appearance (3.Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (453) Google Scholar), which will be referred to here as endosomal carrier vesicles/multivesicular bodies (ECV/MVBs).1 Once formed on early endosomal membranes, ECV/MVBs move toward late endosome, and this movement depends on intact microtubules and motor proteins (3.Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (453) Google Scholar, 4.Aniento F. Emans N. Griffiths G. Gruenberg J. J. Cell Biol. 1993; 123: 1373-1388Crossref PubMed Scopus (366) Google Scholar, 5.Bomsel M. Parton R. Kuznetsov S.A. Schroer T.A. Gruenberg J. Cell. 1990; 62: 719-731Abstract Full Text PDF PubMed Scopus (226) Google Scholar). Eventually, ECVs dock onto and fuse with late endosomes, in a process which depends on α soluble N-ethylmaleimide sensitive factor (NSF) attachment protein, NSF, and perhaps another member of the triple A ATPase family (6.Robinson L. Aniento F. Gruenberg J. J. Cell Sci. 1997; 110: 2079-2087Crossref PubMed Google Scholar), as well as presumably the small GTPase rab7 (7.Feng Y. Press B. Wandinger-Ness A. J. Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (529) Google Scholar). In vivo and in vitro studies have shown that ECV/MVB formation on early endosomes depends on some, but not all, components of the COP-I coat complex (8.Aniento F. Gu F. Parton R. Gruenberg J. J. Cell Biol. 1996; 133: 29-41Crossref PubMed Scopus (314) Google Scholar, 9.Gu F. Aniento F. Parton R. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (138) Google Scholar, 10.Whitney J.A. Gomez M. Sheff D. Kreis T.E. Mellman I. Cell. 1995; 83: 703-713Abstract Full Text PDF PubMed Scopus (265) Google Scholar), which is known to be also involved in the early secretory pathway (11.Lowe M. Kreis T.E. Biochim. Biophys. Acta. 1998; 1404: 53-66Crossref PubMed Scopus (81) Google Scholar). Recent studies, in fact, indicated that endosomal COPs contribute to the down-regulation of the Nef-CD4 complex, via direct interactions between Nef and βCOP (12.Piguet V. Gu F. Foti M. Demaurex N. Gruenberg J. Carpentier J.-L. Trono D. Cell. 1999; 97: 63-73Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Similarly, the AP3 adaptor complex also appears to be involved in more than one pathway, namely transport toward late endosomes/lysosomes (13.Le Borgne R. Hoflack B. Curr. Opin. Cell Biol. 1998; 10: 499-503Crossref PubMed Scopus (91) Google Scholar) and synaptic vesicle formation (14.Faundez V. Horng J.T. Kelly R.B. Cell. 1998; 93: 423-432Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In addition to endosomal COPs, ECV/MVB formation also depends on the acidification properties of early endosomes (15.Clague M. Urbé S. Aniento F. Gruenberg J. J. Biol. Chem. 1994; 269: 21-24Abstract Full Text PDF PubMed Google Scholar). These pH- and COP-dependent processes are related functionally and biochemically, since COP association to endosomal membranes, but not to biosynthetic membranes, is itself pH-sensitive (8.Aniento F. Gu F. Parton R. Gruenberg J. J. Cell Biol. 1996; 133: 29-41Crossref PubMed Scopus (314) Google Scholar, 9.Gu F. Aniento F. Parton R. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (138) Google Scholar). These observations lead us to propose that a transmembrane pH-sensor regulates COP association to endosomes, thereby signaling the onset of the degradation pathway on early endosomal membranes. In the present paper, we have further dissected the molecular process which regulates membrane association of endosomal COPs. Our data show that the small GTP-binding protein ARF1 is required for COP recruitment onto endosomes, and for ECV/MVB biogenesis from donor early endosomal membranes in vitro. We find that PLD and phosphatidic acid are not involved in this process, indicating that ARF1 does not act via PLD on early endosomal membranes, in contrast to biosynthetic COPs (16.Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (328) Google Scholar). Moreover, our data show that ARF1 recruitment onto endosomes depends on the acidic lumenal pH, in agreement with previous studies on ARF binding to microsomes (17.Zeuzem S. Feick P. Zimmermann P. Haase W. Kahn R.A. Schulz I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6619-6623Crossref PubMed Scopus (76) Google Scholar), and that this mechanism accounts for the pH dependence of endosomal COPs association to membranes. Our data thus show that ARF1 mediates COP binding to endosomes and ECV/MVB biogenesis, in a pH-dependent, but PLD-independent, process. ARF1 thus appears to act as the cytosolic component relaying lumenal pH variations to endosomal COPs during ECV/MVB biogenesis. We thank Toshihide Kobayashi for fruitful discussions and help with lipid analysis, Michèle Comte for expertise in protein purification and Marie-Hélène Beuchat for expert technical assistance. We also thank Gisou van der Goot and Julien Fauré for critical reading of the manuscript and all members of the group for suggestions and discussions. We are also very grateful for the generous gift of Actinomadura PLD from Meito Sangyo Company, Tokyo, Japan.
Gu et al. (Wed,) studied this question.