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Molecular Mechanisms in Exocytosis and Endocytosis

Role of neuronal Ca2+-sensor proteins in Golgi–cell-surface membrane traffic Marina Mikhaylova1 , Pasham Parameshwar Reddy and Michael R. Kreutz Project Group Neuroplasticity, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany

Abstract The regulated local synthesis of PtdIns4P and PtdIns(4,5)P2 is crucial for TGN (trans-Golgi network)–plasma membrane trafficking. The activity of PI4Kβ (phosphoinositide 4-kinase IIIβ) at the Golgi membrane is a first mandatory step in this process. In addition to PI4Kβ activity, elevated Ca2+ levels are also needed for the exit of vesicles from the TGN. The reason for this Ca2+ requirement is at present unclear. In the present review, we discuss the role of neuronal Ca2+ -sensor proteins in the regulation of PI4Kβ and suggest that this regulation might impose a need for elevated Ca2+ levels for a late step of vesicle assembly.

Introduction Role of Ca2+ in vesicle formation and trafficking at the Golgi apparatus Many studies have shown that Ca2+ signalling plays a crucial role in Golgi function [1,2]. The Golgi by itself is a Ca2+ store that contains release and sequestration apparatus involved in the generation of Ca2+ transients with apparently slow as well as fast kinetics [1–3]. Instrumental for these Ca2+ gradients are two types of Ca2+ -ATPases, namely a sarcoplasmic reticulum/ER (endoplasmic reticulum) ATPase isoform and a thapsigargin-insensitive, secretory pathway Ca2+ -ATPase. Ca2+ release is triggered by the production of Ins(1,4,5)P3 and its corresponding receptors on the Golgi. Moreover, Ca2+ imaging studies have revealed that Ca2+ concentrations are particularly high in the Golgi region [4] and Ca2+ transients are probably confined to Golgi subcompartments. Several studies have shown that Ca2+ regulates the passage of proteins along the secretory pathway. Taken together, these reports suggest that both Ca2+ -dependent inhibition and stimulation of vesicular transport exist between different compartments of the secretory pathway ([1]; Figure 1). Most importantly, in previous work it has been established that the anterograde-directed exit of vesicles from the Golgi apparatus crucially requires Ca2+ [3] and it is believed that local elevations in free Ca2+ levels play a role in a late step of Golgi secretory function (Figure 1). However, the underlying mechanisms are currently unknown.

Key words: caldendrin, calneuron 1, Golgi, neuronal calcium-sensor protein-1 (NCS-1), phosphoinositide 4-kinase IIIβ (PI4Kβ). Abbreviations used: ER, endoplasmic reticulum; hGH, human growth hormone; NCS, neuronal Ca2+ -sensor protein; PI4Kβ, phosphoinositide 4-kinase IIIβ; PTV, Piccolo–Bassoon transport vesicle; TGN, trans-Golgi network. 1 To whom correspondence should be addressed (email [email protected]).

Biochem. Soc. Trans. (2010) 38, 177–180; doi:10.1042/BST0380177

Budding of vesicles from the TGN (trans-Golgi network) crucially requires PI4Kβ (phosphoinositide 4-kinase IIIβ) activity In addition, the regulated local synthesis of PtdIns4P and PtdIns(4,5)P2 is crucial for TGN–plasma membrane trafficking and the activity of PI4Kβ at the Golgi membrane is a first mandatory step in this process [5–7]. Interestingly, a number of studies have shown that the enzymatic activity of PI4Kβ is regulated by Ca2+ via an interaction with Frequenin/NCS-1 (neuronal Ca2+ sensor protein-1; [8–13]). According to these studies, NCS1 associates with PI4Kβ already in resting conditions, whereas an increase in Ca2+ levels leads to increased enzyme activity due to a Ca2+ -induced conformational change in NCS-1. NCS proteins play multiple and divergent roles in neuronal signalling. Members of this family closely resemble the structure of their common ancestor calmodulin with four EF-hand Ca2+ -binding motifs. Despite their relatively high degree of similarity, NCS proteins are thought to serve highly specialized functions in neurons. It is generally believed that the specificity with respect to their target interactions is brought about by the following: (i) a restricted subcellular localization, (ii) differences in Ca2+ -binding affinities, or (iii) modifications of their EF-hand structure that might provide a unique interface for protein interactions [14–16]. Two novel members of this family are calneurons 1 and 2. Both proteins are highly homologous with each other and are closely related to the caldendrin/CaBP (Ca2+ -binding protein) subfamily of Ca2+ sensors [14–19]. In recent years, we have characterized these two proteins in more detail. We found that calneurons are highly abundant at the Golgi in neurons and that their Golgi association is much more prominent than those of other Ca2+ -sensor proteins such as caldendrin and NCS-1 [20]. Calneurons associate, like NCS1, with PI4Kβ in a Ca2+ -independent manner; at low Ca2+ levels, PI4Kβ is preferentially associated with calneurons,  C The

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Figure 1 Ca2+ -dependent steps in vesicle formation and trafficking Proteins and lipids are initially synthesized in the ER. Export from the ER defines the first step of the pathway and is mediated by the recruitment of COPII (coatamer protein II) protein coats and the subsequent budding of COPII-coated vesicles. The coat proteins are responsible for the initial sorting and the preferential recruitment of cargo and exclusion of resident ER proteins. Budded COPII vesicles fuse with each other and with already fused vesicles to form the polymorphic tubular structures, VTCs (vesicular tubular clusters). This step was shown to be Ca2+ -independent. VTCs integrate the anterograde ER-to-Golgi complex and the retrograde recycling transport pathways. During their limited life span, VTCs undergo maturation by the selective recycling and acquisition of specific proteins and the sequential exchange of specific molecules. For instance, COPII coats exchange for COPI (coatamer protein I) coats on VTCs. It has been demonstrated that association of COPI coats on the vesicles targeted back to ER is Ca2+ -dependent. VTCs accumulate in the peri-Golgi region and are connected to the cis-Golgi network. Intra-Golgi trafficking is regulated by Ca2+ via calmodulin [27]. Cargos subsequently progress through the stack to the TGN, where they are packaged and sent to the required destination. This late step in the TGN is also sensitive to Ca2+ , probably because of a Ca2+ -dependent regulation of one of the key enzymes, namely PI4Kβ, which phosphorylates PtdIns at position 4 and produces PtdIns4P. Lipid rafts enriched in PtdIns4P are thought to mediate clathrin-independent vesicle formation and have been proposed to participate in sorting at the TGN. Finally, trafficking of vesicles that bud off the TGN is Ca2+ -independent.

whereas high Ca2+ levels favour binding of NCS-1 [20]. Stunningly, and in sharp contrast with the activating role of NCS-1, calneurons strongly inhibit PI4Kβ activity in an enzyme activity assay, with markedly attenuated PI4Kβ activity at low to medium Ca2+ levels [20]. Quantification of PtdIns4P levels in COS-7 cells revealed that overexpression of both calneurons significantly reduced PtdIns4P production, indicating that calneurons also inhibit PI4Kβ activity in vivo [20].  C The

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Inhibition and activation of PI4Kβ by NCS proteins regulates Golgi–plasma membrane trafficking In accord with another recent study [21], overexpression experiments in primary neuronal cultures demonstrated that calneurons accumulate at the TGN and not at the cis-Golgi [20]. This resembles the localization of the endogenous proteins and we found an almost complete co-localization with PI4Kβ [20]. Interestingly, the TGN was significantly enlarged after calneuron overexpression. To assess the functional consequences of the calneuron–PI4Kβ interaction for TGN–plasma membrane trafficking in vivo, we examined the effects of calneuron overexpression on ATP-driven hGH (human growth hormone) release from transfected PC12 cells, a cell line in which heterologously expressed calneurons also prominently associate with the Golgi. Previous work has shown that PI4Kβ is crucially involved in the genesis of secretory vesicles in this assay and that their Golgi-trafficking is enhanced by NCS-1 overexpression [12,13,22,23]. For calneuron-transfected cells, hGH release after stimulation was significantly lower and was clearly reduced below the basal release levels observed with the same constructs. Thus, in line with the results of the enzyme activity assay, overexpression of calneurons inhibits Ca2+ -evoked exocytosis in vivo. Synaptophysin is a vesicular component that has to pass via the Golgi to enter axons. We found that overexpression of calneuron-1 inhibits the exit of synaptophysin-positive vesicles from the TGN, whereas the opposite is found after knockdown of calneuron-1 [20]. Most importantly, the application of the Ca2+ -chelating agent BAPTA [bis-(oaminophenoxy)ethane-N,N,N ,N -tetra-acetic acid], which clearly blocks vesicle trafficking from the Golgi in controls, has only a minor effect after RNAi (RNA interference) knockdown of calneuron-1 in primary neurons [20]. We could also demonstrate that calneurons negatively regulate the trafficking of PTVs (Piccolo–Bassoon transport vesicles). PTVs are large dense core vesicles that carry a set of proteins functionally coupled to synaptic vesicle exocytosis and that are thought to be essential for the formation of the presynaptic active zone during development [24,25]. Overexpression of calneurons at the Golgi reduces the number of PTVs in axons and, concomitantly, significantly enlarges the size of the TGN due to a build-up of vesicle proteins [20]. A protein knockdown of calneurons has again the opposite effect. Thus calneurons might provide a Ca2+ threshold for the exit of vesicles from the Golgi in neurons [20].

Competitive binding of calneurons and NCS-1 to PI4Kβ is regulated by Mg2+ These results suggest the existence of a molecular switch in the association of the three proteins with a dominant regulatory role of calneurons at low to medium Ca2+ levels, which is counteracted by NCS-1 at higher Ca2+ levels. However, the Ca2+ -binding affinities of calneuron-1 and NCS-1 are reportedly very similar. We could solve this puzzle by

Molecular Mechanisms in Exocytosis and Endocytosis

Figure 2 Calneurons and NCS-1 provide a Ca2+ -dependent molecular switch between inhibition and activation of PI4Kβ at Golgi membranes At resting Ca2+ concentrations, calneurons (Caln-1) are tightly bound to PI4Kβ and will inhibit PI4Kβ activity (upper panel). This results in an inhibition of Golgi trafficking and the budding of vesicles (upper panel). An increase in Ca2+ concentrations at the Golgi will favour binding of NCS-1 and calneurons are released from the PI4Kβ complex (lower panel). NCS-1 will stimulate the enzymatic activity of PI4Kβ and Golgi trafficking via a 3–4-fold increase in PtdIns4P [PI(4)P] and PtdIns(4,5)P2 production (lower panel). PI-phosphatidylinositol.

deduced that the switch from calneuron to NCS-1 binding can induce a 3–4-fold increase in PtdIns4P production, which represents a major effect for the availability of this rare phospholipid (Figure 2). Taken together, this assigns a cellular function to calneurons, which add an important regulatory mechanism for stimulus-dependent dynamics in TGN–plasma membrane trafficking.

Funding This work was supported by research grants from the Bundesministerium fur ¨ Bildung und Forschung (Federal Ministry of Education and Research), Deutsche Forschungsgemeinschaft, Centre for Behavioural Brain Sciences and the Schram Foundation. Work on this paper was directly supported by a Department of Science and Technology Deutscher Akademischer Austauschdienst (DST-DAAD) scholar exchange grant and a Bundesministerium fur ¨ Bildung und Forschung Department of Biotechnology (BMBF-DBT) international co-operation grant awarded to M.R.K.

References

showing that Mg2+ binding of NCS-1 drastically reduces its Ca2+ -binding affinity [20,26]. In sharp contrast with NCS-1, Mg2+ does not bind at physiologically relevant concentrations to calneurons [20]. Irrespective of Mg2+ levels, Ca2+ associates at Ca2+ -specific regulatory sites with apparent global affinities of approx. 180 nM (calneuron1; [17,20]) and 230 nM (calneuron-2; [20]). It is important to note that free Mg2+ levels in a cell will render NCS-1 always in a Mg2+ -bound state. This in turn will promote the reversibility to a Ca2+ -free state since the resting Ca2+ levels of approx. 100 nM would otherwise keep NCS-1 always in a Ca2+ -bound state. Calneurons, however, have in contrast with NCS-1 a very narrow dynamic range of Ca2+ -induced unfolding with much less reversibility to the Ca2+ -free state, and this explains why they dominate in the regulation of PI4Kβ activity at low to medium Ca2+ concentrations. Our results suggest that calneurons operate as a filter that suppresses PI4Kβ activity at submaximal amplitudes of Golgi Ca2+ transients and thereby provides a tonic inhibition that is only released under conditions of sustained Ca2+ release (Figure 2). The opposing roles of calneurons and NCS-1 in PI4Kβ-mediated PtdIns4P production suggest a scenario with only two discrete states and little fine-tuning of enzyme activity between both states (Figure 2). Importantly, it can be

1 Dolman, N.J. and Tepikin, A.V. (2006) Calcium gradients and the Golgi. Cell Calcium 40, 505–512 2 Hay, J.C. (2007) Calcium: a fundamental regulator of intracellular membrane fusion? EMBO Rep. 8, 236–240 3 Chen, J.L., Ahluwalia, J.P. and Stamnes, M. (2002) Selective effects of calcium chelators on anterograde and retrograde protein transport in the cell. J. Biol. Chem. 277, 35682–35687 4 Pinton, P., Pozzan, T. and Rizzuto, R. (1998) The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum. EMBO J. 17, 5298–5308 5 Michell, R.H. (2008) Inositol derivatives: evolution and functions. Nat. Rev. Mol. Cell Biol. 9, 151–161 6 Balla, A. and Balla, T. (2006) Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol. 16, 352–361 7 De Matteis, M.A. and Luini, A. (2008) Exiting the Golgi complex. Nat. Rev. Mol. Cell Biol. 9, 273–284 8 Hendricks, K.B., Wang, B.Q., Schnieders, E.A. and Thorner, J. (1999) Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase. Nat. Cell Biol. 1, 234–241 9 Zhao, X., Varnai, ´ P., Tuymetova, G., Balla, A., Toth, ´ Z.E., Oker-Blom, C., Roder, J., Jeromin, A. and Balla, T. (2001) Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase β stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells. J. Biol. Chem. 276, 40183–40189 10 Huttner, I.G., Strahl, T., Osawa, M., King, D.S., Ames, J.B. and Thorner, J. (2003) Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1. J. Biol. Chem. 278, 4862–4874 11 Taverna, E., Francolini, M., Jeromin, A., Hilfiker, S., Roder, J. and Rosa, P. (2002) Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase β interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis. J. Cell Sci. 115, 3909–3922 12 De Barry, J., Janoshazi, A., Dupont, J.L., Procksch, O., Chasserot-Golaz, S., Jeromin, A. and Vitale, N (2006) Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-β interaction in regulated exocytosis of PC12 cells. J. Biol. Chem. 281, 18098–18111 13 Haynes, L.P., Thomas, G.M. and Burgoyne, R.D. (2005) Interaction of neuronal calcium sensor-1 and ADP-ribosylation factor 1 allows bidirectional control of phosphatidylinositol 4-kinase β trans-Golgi network–plasma membrane traffic. J. Biol. Chem. 280, 6047–6054 14 Burgoyne, R.D. (2007) Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat. Rev. Neurosci. 8, 182–193 15 Burgoyne, R.D., O’Callaghan, D.W., Hasdemir, B., Haynes, L.P. and Tepikin, A.V. (2004) Neuronal Ca2+ -sensor proteins: multitalented regulators of neuronal function. Trends Neurosci. 27, 203–209 16 Seidenbecher, C.I., Reissner, C. and Kreutz, M.R. (2002) Caldendrins in the inner retina. Adv. Exp. Med. Biol. 514, 451–463  C The

C 2010 Biochemical Society Authors Journal compilation 

179

180

Biochemical Society Transactions (2010) Volume 38, part 1

17 Mikhaylova, M., Sharma, Y., Reissner, C., Nagel, F., Aravind, P., Rajini, B., Smalla, K.H., Gundelfinger, E.D. and Kreutz, M.R. (2006) Neuronal Ca2 + signaling via caldendrin and calneurons. Biophys. Biochem. Acta 1763, 1229–1237 18 Wu, Y.Q., Lin, X., Liu, C.M., Jamrich, M. and Shaffer, L.G. (2001) Identification of a human brain-specific gene, calneuron 1, a new member of the calmodulin superfamily. Mol. Genet. Metab. 72, 343–350 19 Seidenbecher, C.I., Langnaese, K., Sanmart´ı-Vila, L., Boeckers, T.M., Smalla, K.H., Sabel, B.A., Garner, C.C., Gundelfinger, E.D. and Kreutz, M.R. (1998) Caldendrin, a novel neuronal calcium-binding protein confined to the somato-dendritic compartment. J. Biol. Chem. 273, 21324–21334 20 Mikhaylova, M., Reddy, P.P., Munsch, T., Landgraf, P., Suman, S.K., Smalla, K.H., Gundelfinger, E.D., Sharma, Y. and Kreutz, M.R. (2009) Calneurons provide a calcium-threshold for trans-Golgi network to plasma membrane trafficking. Proc. Natl. Acad. Sci. U.S.A. 106, 9093–9098 21 McCue, H.V., Burgoyne, R.D. and Haynes, L.P. (2009) Membrane targeting of the EF-hand containing calcium-sensing proteins CaBP7 and CaBP8. Biochem. Biophys. Res. Commun. 380, 825–831 22 Koizumi, S., Rosa, P., Willars, G.B., Challiss, R.A., Taverna, E., Francolini, M., Bootman, M.D., Lipp, P., Inoue, K., Roder, J. and Jeromin, A. (2002) Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells. J. Biol. Chem. 277, 30315–30324

 C The

C 2010 Biochemical Society Authors Journal compilation 

23 Rajebhosale, M., Greenwood, S., Vidugiriene, J., Jeromin, A. and Hilfiker, S. (2003) Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells. J. Biol. Chem. 278, 6075–6084 24 Zhai, R.G., Vardinon-Friedman, H., Cases-Langhoff, C., Becker, B., Gundelfinger, E.D., Ziv, N.E. and Garner, C.C. (2001) Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29, 131–143 25 Shapira, M., Zhai, R.G., Dresbach, T., Bresler, T., Torres, V.I., Gundelfinger, E.D., Ziv, N.E. and Garner, C.C. (2003) Unitary assembly of presynaptic active zones from Piccolo–Bassoon transport vesicles. Neuron 38, 237–252 26 Aravind, P., Chandra, K., Reddy, P.P., Jeromin, A., Chary, K.V. and Sharma, Y. (2008) Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg2+ modulates Ca2+ binding, Ca2+ -induced conformational changes, and equilibrium unfolding transitions. J. Mol. Biol. 376, 1100–1115 27 Porat, A. and Elazar, Z. (2000) Regulation of intra-Golgi membrane transport by calcium. J. Biol. Chem. 275, 29233–29237

Received 4 April 2009 doi:10.1042/BST0380177