A Regulatory Role for cAMP-dependent Protein Kinase in Protein Traffic along the Exocytic Route

Abstract

The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization. The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization. INTRODUCTIONRecent evidence suggests a regulatory role for signal transduction molecules in vesicle-mediated transport along both the exocytic and endocytic pathways. Several heterotrimeric G proteins have been implicated in different steps of membrane trafficking (1Bomsel M. Mostov K. Mol. Biol. Cell. 1992; 3: 1317-1328Crossref PubMed Scopus (162) Google Scholar, 2Nuoffer C. Balch W.E. Annu. Rev. Biochem. 1994; 63: 949-990Crossref PubMed Scopus (372) Google Scholar). Second messengers and protein kinases have also been shown to influence various transport processes. For instance, cAMP and protein kinase A activity have been reported to stimulate apical directed transcytosis and secretion in epithelial cells (3Eker P. Holm P.K. van Deurs B. Sandving K. J. Biol. Chem. 1994; 269: 18607-18615Abstract Full Text PDF PubMed Google Scholar, 4Hansen S.H. Casanova J.E. J. Cell Biol. 1994; 126: 677-687Crossref PubMed Scopus (117) Google Scholar, 5Takuma T. Ichida T. J. Biol. Chem. 1994; 269: 22124-22128Abstract Full Text PDF PubMed Google Scholar, 6Jilling T. Kirk K.L. J. Biol. Chem. 1996; 271: 4381-4387Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Protein kinase A seems to exert a differential regulatory role in polarized cells. Transport from the trans-Golgi network (TGN) 1The abbreviations used are: TGNtrans-Golgi networkERendoplasmic reticulumVSVvesicular stomatitis virusVSV-GVSV G glycoproteinIBMX3-isobutyl-1-methylxanthineFKforskolindibutyryl-cAMPN6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate8-Br-cAMP8-bromoadenosine-3′,5′-cyclic monophosphateH-89N-[2-(-p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamideSp-cBIMPs5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate Sp isomerendo Hendoglycosidase HNRKnormal rat kidneyPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered saline. to the apical surface but not to the basolateral surface was stimulated by agents that activate protein kinase A (6Jilling T. Kirk K.L. J. Biol. Chem. 1996; 271: 4381-4387Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Pimplikar S.W. Simons K. J. Biol. Chem. 1994; 269: 19054-19059Abstract Full Text PDF PubMed Google Scholar). Phorbol esters and protein kinase C have also been implicated in transport regulation. Association of ADP-ribosylation factor and coatomer proteins to Golgi membranes was increased by treatment with agents that activate protein kinase C (8De Matteis M.A. Santini G. Kahn R.A. Di Tullio G. Luini A. Science. 1993; 364: 818-820Google Scholar). Constitutive transport from the TGN to the cell surface, regulated secretion, apical exocytosis, and transcytosis have all been shown to be modulated by protein kinase C activity (7Pimplikar S.W. Simons K. J. Biol. Chem. 1994; 269: 19054-19059Abstract Full Text PDF PubMed Google Scholar, 9Cardone M.H. Smith B.L. Song W. Mochly-Rosen D. Mostov K.E. J. Cell Biol. 1994; 124: 717-727Crossref PubMed Scopus (93) Google Scholar, 10Akita Y. Ohno S. Yajima Y. Konno Y. Saido T.C. Mizuno K. Chida K. Osada S. Kuroki T. Kawashima S. Suzuki K. J. Biol. Chem. 1994; 269: 4653-4660Abstract Full Text PDF PubMed Google Scholar, 11Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 12Buccione R. Bannykh S. Santone I. Baldassarre M. Facchiano F. Bozzi Y. Di Tullio G. Mironov A. Luini A. De Matteis M.A. J. Biol. Chem. 1996; 271: 3523-3533Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Together, these studies extend previous observations on the requirement for protein phosphorylation in certain transport steps such as protein transfer from the endoplasmic reticulum (ER) to the Golgi complex (13Davidson H.W. McGowan C.H. Balch W.E. J. Cell Biol. 1992; 116: 1343-1355Crossref PubMed Scopus (103) Google Scholar), formation of secretory vesicles from the TGN (14Ohashi M. Huttner W.B. J. Biol. Chem. 1994; 269: 24897-24905Abstract Full Text PDF PubMed Google Scholar), and endosome fusion (15Woodman P.G. Mundy D.I. Cohen P. Warren G. J. Cell Biol. 1992; 116: 331-338Crossref PubMed Scopus (53) Google Scholar). Particular isoforms of both protein kinases A and C have been localized in the ER and the Golgi complex (16Rios R.M. Celati C. Lohmann S.M. Bornens M. Keryer G. EMBO J. 1992; 11: 1723-1731Crossref PubMed Scopus (55) Google Scholar, 17Nigg E.A. Schäfer G. Hilz H. Eppenberger H.M. Cell. 1985; 41: 1039-1051Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 18Goodnight J. Mischak H. Kolch W. Mushinski J.F. J. Biol. Chem. 1995; 270: 9991-10001Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 19Lehel C. Olah Z. Jakab G. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1406-1410Crossref PubMed Scopus (123) Google Scholar). It is unknown, however, in what way these kinases modulate either organelle structure and/or transport processes. Other evidences indicate an essential role for protein kinases in particular transport steps. Transport of hydrolases from the TGN to either the yeast vacuole or mammalian lysosomes requires phosphatidylinositol 3-kinase activity (13Davidson H.W. McGowan C.H. Balch W.E. J. Cell Biol. 1992; 116: 1343-1355Crossref PubMed Scopus (103) Google Scholar, 20Brown W.J. DeWald D.B. Emr S.D. Plutner H. Balch W.E. J. Cell Biol. 1995; 130: 781-796Crossref PubMed Scopus (251) Google Scholar, 21Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Crossref PubMed Scopus (261) Google Scholar). Therefore, the available evidences indicate that different protein kinases regulate membrane trafficking in eukaryotic cells although the importance and extent of such control is unclear at present. In this study we have investigated the role of cAMP and protein kinase A activity on transport along the entire exocytic pathway. We studied the constitutive transfer of an integral membrane glycoprotein, vesicular stomatitis virus (VSV)-G protein, from the ER through the Golgi to the cell surface. The results indicate a stimulatory role for protein kinase A on different steps along this route and emphasize the influence of this activity on Golgi structural organization.DISCUSSIONThe transport machinery responsible for vesicle budding, targeting, and fusion is under regulation. Protein kinases and second messengers seem to be involved in this control. We have studied the role played by cAMP and protein kinase A on protein transport along the exocytic pathway. The results indicate that protein kinase A stimulates several transport steps in this route although the extent of regulation varies. Thus, transport from the TGN to the cell surface was shown to be dependent on activated protein kinase A (Fig. 3, Fig. 4), whereas transport from the ER to the cis-/middle Golgi (Fig. 5) or from the intermediate compartment to the TGN (Fig. 6) was sensitive to but did not require protein kinase A activation. This could reflect the involvement of particular transport molecules in each case. In a similar way, the effects of protein kinase A activation on transport activities can differ from one cell system to another. In Madin-Darbin canine kidney-polarized cells, for example, transport from the ER to the Golgi and from the TGN to the basolateral surface was insensitive to protein kinase A activation, whereas transport to the apical surface was stimulated (7Pimplikar S.W. Simons K. J. Biol. Chem. 1994; 269: 19054-19059Abstract Full Text PDF PubMed Google Scholar). Therefore, it is possible that the protein kinase A substrates relevant to transport processes would be selective proteins required for a particular transfer event rather than molecules involved in multiple transport reactions.Transport from the TGN to the cell surface was blocked by H-89 (Fig. 4B) and stimulated by IBMX + FK treatment (Fig. 9B). The first agent has been shown to specifically inhibit protein kinase A (30Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar), whereas it is commonly accepted that incubation with either IBMX or FK leads to protein kinase A activation by increasing the intracellular level of cAMP. In principle, the increased number of VSV-G molecules detected at the surface of cells chased in the presence of IBMX + FK could result from an inhibitory effect of these agents on protein uptake by endocytosis. However, we used 125I-labeled epidermal growth as a ligand for receptor-mediated endocytosis, and we determined that the internalization rates did not differ significantly between control and treated cells. 2M. Alonso, M. Muñiz, J. Hidalgo, and A. Velasco, unpublished observations. It is then plausible to assume that an increased level of cAMP caused by IBMX + FK incubation stimulate export of VSV-G from the TGN to the cell surface. Similarly, the inhibitory effect of H-89 cannot be explained by a decrease in the cellular levels of ATP necessary for transport (Fig. 1) (36Balch W.E. Elliott M.M. Keller D.S. J. Biol. Chem. 1986; 261: 14681-14689Abstract Full Text PDF PubMed Google Scholar). Instead, the data support a positive regulatory role for protein kinase A in vesicle transport from the TGN to the cell surface in nonpolarized cells. This transport step has been commonly considered to occur constitutively. However, recent reports indicate that it is influenced by different signal transduction molecules (6Jilling T. Kirk K.L. J. Biol. Chem. 1996; 271: 4381-4387Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 12Buccione R. Bannykh S. Santone I. Baldassarre M. Facchiano F. Bozzi Y. Di Tullio G. Mironov A. Luini A. De Matteis M.A. J. Biol. Chem. 1996; 271: 3523-3533Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this context, changes in the intracellular concentration of cAMP and other second messengers could control vesicle formation and/or protein sorting at the TGN by exerting a regulatory role on the protein machinery responsible for these activities.In general results obtained with IBMX and FK were consistent with a stimulatory role of activated protein kinase A on transport. However, these agents also gave rise to important modifications of Golgi structure which could mask their influence on transport activities. Thus, although by immunofluorescence ER to Golgi transport was accelerated in IBMX + FK-treated cells (Fig. 7), acquisition of endo H resistance was hindered in the altered Golgi complex (Fig. 9A). Also, transport out of the Golgi complex was decreased in cells exposed to IBMX and FK for a prolonged time causing retention of VSV-G protein in this organelle (Fig. 7H). For these reasons we believe that both the ability to process glycoproteins and to produce functional transport vesicles were affected in the Golgi complex modified by IBMX and FK treatment. The modifications consisted in intercisternae fusion within the Golgi stacks which gave rise to irregular Golgi compartments and miscompartmentalization of resident Golgi proteins (Fig. 8, A and C). These structural changes were derived from an increase in the intracellular level of cAMP since they were also observed in cells incubated with cholerae toxin (Fig. 8, D-H) or treated with cAMP analogs. Interestingly, an increase in cAMP has been reported to induce alkalinization of the trans-Golgi lumen (37Seksek O. Biwersi J. Verkman A.S. J. Biol. Chem. 1995; 270: 4967-4970Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and to affect protein glycosylation (6Jilling T. Kirk K.L. J. Biol. Chem. 1996; 271: 4381-4387Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 38Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. Al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (422) Google Scholar). In any case, these observations suggest a regulatory role for protein kinase A on the maintenance of the structural organization of the Golgi complex.Protein kinase A is an ubiquitous regulatory enzyme activated by cAMP binding which in turn phosphorylates specific protein substrates. Cell signaling processes that are mediated by protein kinase A begin with the activation of a plasma membrane receptor coupled to heterotrimeric G proteins. These modulate cAMP production by acting on cAMP-phosphodiesterase and adenylate cyclase. It is becoming increasingly evident that a similar signaling pathway controls intracellular membrane trafficking. Several kinds of heterotrimeric G proteins, including Gαs, Gαi, and XLαs proteins, have been detected in intracellular membranes and implicated in vesicular transport along both the exocytic and endocytic routes (1Bomsel M. Mostov K. Mol. Biol. Cell. 1992; 3: 1317-1328Crossref PubMed Scopus (162) Google Scholar, 39Leyte A. Barr F.A. Kehlenbach R.H. Huttner W.B. EMBO J. 1992; 11: 4795-4804Crossref PubMed Scopus (139) Google Scholar, 40Stow J.L. Almeida J.B. d. Narula N. Holtzman E.J. Ercolani L. Ausiello D.A. J. Cell Biol. 1991; 114: 1113-1124Crossref PubMed Scopus (239) Google Scholar, 41Kehlenbach R.H. Matthey J. Huttner W.B. Nature. 1994; 372: 804-809Crossref PubMed Scopus (201) Google Scholar, 42Montmayeur J.-P. Borrelli E. Science. 1994; 263: 95-98Crossref PubMed Scopus (52) Google Scholar, 43Colombo M.I. Mayorga L.S. Nishimoto I. Ross E.M. Stahl P.D. J. Biol. Chem. 1994; 269: 14919-14923Abstract Full Text PDF PubMed Google Scholar). It is postulated that these proteins transmit signals to effectors that could regulate transport in a still undetermined way. While ER- and Golgi-associated protein kinases (17Nigg E.A. Schäfer G. Hilz H. Eppenberger H.M. Cell. 1985; 41: 1039-1051Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 19Lehel C. Olah Z. Jakab G. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1406-1410Crossref PubMed Scopus (123) Google Scholar) are potential downstream effectors, other evidences suggest that second messengers and protein kinases are not necessarily involved (44Lang J. Nishimoto I. Okamoto T. Regazzi R. Kiraly C. Weller U. Wollheim C.B. EMBO J. 1995; 14: 3635-3644Crossref PubMed Scopus (125) Google Scholar). Therefore, it will be important to determine in which way protein kinase A controls transport reactions. INTRODUCTIONRecent evidence suggests a regulatory role for signal transduction molecules in vesicle-mediated transport along both the exocytic and endocytic pathways. Several heterotrimeric G proteins have been implicated in different steps of membrane trafficking (1Bomsel M. Mostov K. Mol. Biol. Cell. 1992; 3: 1317-1328Crossref PubMed Scopus (162) Google Scholar, 2Nuoffer C. Balch W.E. Annu. Rev. Biochem. 1994; 63: 949-990Crossref PubMed Scopus (372) Google Scholar). Second messengers and protein kinases have also been shown to influence various transport processes. For instance, cAMP and protein kinase A activity have been reported to stimulate apical directed transcytosis and secretion in epithelial cells (3Eker P. Holm P.K. van Deurs B. Sandving K. J. Biol. Chem. 1994; 269: 18607-18615Abstract Full Text PDF PubMed Google Scholar, 4Hansen S.H. Casanova J.E. J. Cell Biol. 1994; 126: 677-687Crossref PubMed Scopus (117) Google Scholar, 5Takuma T. Ichida T. J. Biol. Chem. 1994; 269: 22124-22128Abstract Full Text PDF PubMed Google Scholar, 6Jilling T. Kirk K.L. J. Biol. Chem. 1996; 271: 4381-4387Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Protein kinase A seems to exert a differential regulatory role in polarized cells. Transport from the trans-Golgi network (TGN) 1The abbreviations used are: TGNtrans-Golgi networkERendoplasmic reticulumVSVvesicular stomatitis virusVSV-GVSV G glycoproteinIBMX3-isobutyl-1-methylxanthineFKforskolindibutyryl-cAMPN6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate8-Br-cAMP8-bromoadenosine-3′,5′-cyclic monophosphateH-89N-[2-(-p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamideSp-cBIMPs5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate Sp isomerendo Hendoglycosidase HNRKnormal rat kidneyPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered saline. to the apical surface but not to the basolateral surface was stimulated by agents that activate protein kinase A (6Jilling T. Kirk K.L. J. Biol. Chem. 1996; 271: 4381-4387Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Pimplikar S.W. Simons K. J. Biol. Chem. 1994; 269: 19054-19059Abstract Full Text PDF PubMed Google Scholar). Phorbol esters and protein kinase C have also been implicated in transport regulation. Association of ADP-ribosylation factor and coatomer proteins to Golgi membranes was increased by treatment with agents that activate protein kinase C (8De Matteis M.A. Santini G. Kahn R.A. Di Tullio G. Luini A. Science. 1993; 364: 818-820Google Scholar). Constitutive transport from the TGN to the cell surface, regulated secretion, apical exocytosis, and transcytosis have all been shown to be modulated by protein kinase C activity (7Pimplikar S.W. Simons K. J. Biol. Chem. 1994; 269: 19054-19059Abstract Full Text PDF PubMed Google Scholar, 9Cardone M.H. Smith B.L. Song W. Mochly-Rosen D. Mostov K.E. J. Cell Biol. 1994; 124: 717-727Crossref PubMed Scopus (93) Google Scholar, 10Akita Y. Ohno S. Yajima Y. Konno Y. Saido T.C. Mizuno K. Chida K. Osada S. Kuroki T. Kawashima S. Suzuki K. J. Biol. Chem. 1994; 269: 4653-4660Abstract Full Text PDF PubMed Google Scholar, 11Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 12Buccione R. Bannykh S. Santone I. Baldassarre M. Facchiano F. Bozzi Y. Di Tullio G. Mironov A. Luini A. De Matteis M.A. J. Biol. Chem. 1996; 271: 3523-3533Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Together, these studies extend previous observations on the requirement for protein phosphorylation in certain transport steps such as protein transfer from the endoplasmic reticulum (ER) to the Golgi complex (13Davidson H.W. McGowan C.H. Balch W.E. J. Cell Biol. 1992; 116: 1343-1355Crossref PubMed Scopus (103) Google Scholar), formation of secretory vesicles from the TGN (14Ohashi M. Huttner W.B. J. Biol. Chem. 1994; 269: 24897-24905Abstract Full Text PDF PubMed Google Scholar), and endosome fusion (15Woodman P.G. Mundy D.I. Cohen P. Warren G. J. Cell Biol. 1992; 116: 331-338Crossref PubMed Scopus (53) Google Scholar). Particular isoforms of both protein kinases A and C have been localized in the ER and the Golgi complex (16Rios R.M. Celati C. Lohmann S.M. Bornens M. Keryer G. EMBO J. 1992; 11: 1723-1731Crossref PubMed Scopus (55) Google Scholar, 17Nigg E.A. Schäfer G. Hilz H. Eppenberger H.M. Cell. 1985; 41: 1039-1051Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 18Goodnight J. Mischak H. Kolch W. Mushinski J.F. J. Biol. Chem. 1995; 270: 9991-10001Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 19Lehel C. Olah Z. Jakab G. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1406-1410Crossref PubMed Scopus (123) Google Scholar). It is unknown, however, in what way these kinases modulate either organelle structure and/or transport processes. Other evidences indicate an essential role for protein kinases in particular transport steps. Transport of hydrolases from the TGN to either the yeast vacuole or mammalian lysosomes requires phosphatidylinositol 3-kinase activity (13Davidson H.W. McGowan C.H. Balch W.E. J. Cell Biol. 1992; 116: 1343-1355Crossref PubMed Scopus (103) Google Scholar, 20Brown W.J. DeWald D.B. Emr S.D. Plutner H. Balch W.E. J. Cell Biol. 1995; 130: 781-796Crossref PubMed Scopus (251) Google Scholar, 21Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Crossref PubMed Scopus (261) Google Scholar). Therefore, the available evidences indicate that different protein kinases regulate membrane trafficking in eukaryotic cells although the importance and extent of such control is unclear at present. In this study we have investigated the role of cAMP and protein kinase A activity on transport along the entire exocytic pathway. We studied the constitutive transfer of an integral membrane glycoprotein, vesicular stomatitis virus (VSV)-G protein, from the ER through the Golgi to the cell surface. The results indicate a stimulatory role for protein kinase A on different steps along this route and emphasize the influence of this activity on Golgi structural organization.