Golgi structural stability and biogenesis depend on associated PKA activity

3764 Research Article Introduction The unique compartmentalized organization of the Golgi complex in mammalian cells is thought to provide optimal structural support for the ordered, sequential processing of molecules along the exocytic pathway. The main part of the Golgi consists of stacks of flat cisternae whose membranes are crowded with enzymes and receptors. During their journey across the stacked cisternae, newly synthesized proteins and lipids are subjected to extensive modifications and are finally sorted for delivery to different cellular destinations (Dunphy and Rothman, 1985). Membrane-associated coiled-coil proteins form a crosslinked matrix that is responsible of cisternae stacking (Barr and Short, 2003). In addition, the stacks of cisternae are laterally linked by tubular connections and this gives the Golgi complex the appearance of a continuous juxtanuclear ribbon (Ladinsky et al., 1999). Golgi structural integrity depends on export from the endoplasmic reticulum (ER). During their life span, Golgi transmembrane proteins such as glycosyltransferases and receptors continuously travel to the ER and re-emerge to enter the Golgi again. The extent of ER recycling varies among proteins. Receptors and components of the transport machinery continuously cycle between the ER and the Golgi as required by their functional activity (Lee et al., 2004). Processing enzymes, however, constitutively cycle at a low rate (Storrie et al., 1998). The functional significance of their cycling is not so obvious but it may be important for both Golgi remodeling during interphase and partitioning of this organelle during mitosis (Altan-Bonnet et al., 2004; Altan-Bonnet et al., 2006; Ward et al., 2001). The existence of this pathway, however, is highlighted by experiments in which ER export is blocked by either pharmacological (i.e. brefeldin A, BFA) treatment or interfering with the assembly of the export machinery (Lippincott-Schwartz et al., 1989; Ward et al., 2001). In both cases, Golgi components redistribute to the ER as expected from the continuous Golgi-ER retrograde traffic. Export from the ER occurs at specialized subdomains known as ER-export sites whose organization depends on the association of the COPII coat transport complex to the ER membrane (Bannykh et al., 1996). They are considered to play an essential role in Golgi reassembly from the ER (Bevis et al., 2003). Thus, both Golgi transmembrane proteins and peripherally associated proteins such as components of the Golgi matrix become concentrated at the ER exit sites during Golgi outgrowth from the ER (Puri and Linstedt, 2003; Ward et al., 2001). In addition to its established role in protein processing and sorting, the Golgi complex is increasingly recognized as a center for signal reception and transmission. This view considers the Golgi complex as a structural scaffold where different signal transduction pathways converge and are propagated to effectors located elsewhere in the cell (Donaldson and Lippincott-Schwartz, 2000). It would explain the presence of a significant number of regulatory molecules associated with the Golgi membranes such as G proteins, phospholipid and protein kinases, phospholipases, cytoskeleton motor proteins, etc. (reviewed by Altan-Bonnet The mammalian Golgi complex consists of stacks of cisternae linked laterally into a continuous perinuclear ribbon structure. Protein kinase A is stably associated with the Golgi complex during interphase. To analyze its role in Golgi structural maintenance cells were depleted of protein kinase A regulatory subunits using small interfering RNAs. Under these conditions, the catalytic subunits redistributed to the cytosol and the entire Golgi complex underwent disassembly into multiple juxtanuclear fragments. A similar effect took place following pharmacological inhibition or redistribution of the complete holoenzyme to the cytosol. Golgi fragments maintained their polarization and competence for anterograde protein trafficking. By electron microscopy, they were identified as whorl-like structures composed of concentrically arrayed cisternae. To test a possible role of protein kinase A in Golgi biogenesis we analyzed its involvement during Golgi reassembly from the endoplasmic reticulum. In cells incubated with protein kinase A inhibitors, Golgi reconstruction was arrested at a late step of the reassembly process. This is consistent with the stage of enzyme recruitment from cytosol to emerging Golgi membranes during the reassembly process. We conclude that protein kinase A activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from separated membrane stacks. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/18/3764/DC1 Key words: Golgi complex, Protein kinase A, AKAP Summary Golgi structural stability and biogenesis depend on associated PKA activity Eloy Bejarano*, Margarita Cabrera*, Lucia Vega, Josefina Hidalgo and Angel Velasco‡ Department of Cell Biology, Faculty of Biology, University of Seville, Avd. Reina Mercedes s/n, 41012 Seville, Spain *These authors contributed equally to this work ‡Author for correspondence (e-mail: av[email protected]) Accepted 20 June 2006 Journal of Cell Science 119, 3764-3775 Published by The Company of Biologists 2006 doi:10.1242/jcs.03146 Journal of Cell Science 3765 Golgi organization requires PKA activity et al., 2004). Protein kinase A (PKA) is a well-known constituent of the Golgi complex (Nigg et al., 1985b). This tetrameric enzyme is associated with the cytosolic surface of Golgi membranes through the interaction of the dimer of regulatory RII␣subunits with a putative anchor protein, whereas the two catalytic C␣subunits are bound to RII␣. Previous work has revealed that Golgi-associated PKA activity regulates protein transport, particularly the formation of membrane intermediates for the anterograde and retrograde transport routes (Cabrera et al., 2003; Martín et al., 2000; Muñiz et al., 1996; Muñiz et al., 1997). Nevertheless, the functional significance of PKA association with the Golgi membranes is mostly unknown. For instance, although pioneer work by Nigg et al. (Nigg et al., 1985a) indicated that Golgiassociated PKA participates in the control of gene expression, its involvement in a particular signal transduction pathway has not yet been reported. Also, the role of PKA association in Golgi structural organization and biogenesis has not been investigated. In this study, we have examined the effects derived from PKA enzymatic inhibition or displacement on Golgi structure. Our data indicate that Golgi maintenance depends on the associated PKA activity. Furthermore, we report that PKA is recruited from the cytosol during Golgi reassembly from the ER and that PKA inhibition interferes with Golgi reconstruction. Fig. 1. Kinetics of RII␣binding to and dissociation from the Golgi. Cells stably expressing RII␣-GFP were photobleached with a high-intensity laser light and then the recovery of fluorescence was monitored by taking images at the indicated time points (in minutes). (A) The Golgi complex (outlined region) was subjected to a single bleach and recovery was analyzed by FRAP. Note that the centrosome was excluded from the bleached area. (B) The cytoplasm (area between the two encircled structures) was bleached repeatedly (every 4 minutes) to analyze RII␣- GFP loss from the Golgi by FLIP. (C) Time-course of the evolution of Golgi-associated fluorescence during both FRAP and FLIP. Values are the mean ± s.e.m. of three different determinations. Journal of Cell Science 3766 Results Dynamics of Golgi-associated PKA Living HeLa cells stably expressing RII␣-GFP were used in photobleaching experiments to determine the dynamics of Golgi-associated PKA. In these cells, RII␣-GFP was concentrated in the Golgi complex and the centrosome, and also dispersed throughout the cytoplasm, mirroring the localization pattern of the endogenous protein (Martin et al., 1999). PKA recruitment from cytosol to the Golgi membranes was analyzed by FRAP (Fig. 1A). Golgi fluorescence recovery occurred with a half-time of 3.65 minutes. This is in contrast with FRAP data obtained with ␥-COP-YFP. Under similar experimental conditions this coat protein associated rapidly (half-time 35.47 seconds) to the surface of the Golgi membranes. This is in agreement with previous reports on coatomer recruitment (Presley et al., 2002). In the case of RII␣- GFP, Golgi fluorescence recovery was rather incomplete because only 73-80% of the initial, prebleach value was reached after prolonged incubations (>1 hour) (Fig. 1C). This suggested the existence of a significant (estimated to be 26%) immobile fraction of this protein permanently associated with the Golgi membranes. Furthermore, FLIP determinations also supported this view (Fig. 1B). PKA dissociation from the Golgi to the cytosol took place with a half-time of 21.3±0.3 minutes (Fig. 1C), which is also a high value for a peripheral Golgi protein. Taken together, these measurements indicate that many of the PKA molecules associated with the Golgi complex are locked at this location and did not exchange rapidly with the population of molecules that diffuse freely throughout the cytosol. Effect of RII␣depletion on Golgi organization To examine the effect of PKA displacement on Golgi structural organization we incubated cells with siRNA designed to specifically inhibit RII␣biosynthesis. We used two siRNAs corresponding to non-overlapping sequences in RII␣with similar efficiency in both cases. Results obtained with one of them (siRNA1) are shown in Fig. 2 and subsequent figures. Journal of Cell Science 119 (18) Golgi depletion was effective after 72-80 hours and affected 70-90% of the cells as judged by immunofluorescence detection (Fig. 2A). Accordingly, during this treatment the total amount of RII␣decreased to 11% of control, untreated cells without significant decrease in the amount of the catalytic C␣ subunit (Fig. 2B). In comparison to the Golgi complex, it took longer, at least 96 hours, for the centrosome to be completely deprived of RII␣. In RII␣-depleted cells, the C␣subunit was dissociated from the Golgi and appeared dispersed throughout the cytoplasm (Fig. 2A). Indeed, the amount of C␣associated with total microsomal membranes was reduced compared with untreated cells (Fig. 2C). No depletion was obtained when cells were incubated with a control siRNA designed to target the rodent form of RII␣(Fig. 2A). Therefore, incubation with siRNA specific for human RII␣induced the redistribution of PKA catalytic activity from the Golgi complex to cytosol. To evaluate the effect of RII␣depletion on Golgi organization we used cells stably expressing a truncated version of galactosyltransferase tagged with CFP. In preliminary experiments, this construct was shown to exhibit a widely extended localization pattern across the Golgi stack. Thus, significant colocalization was observed with endogenous protein markers from both the cis- (giantin, GM130) and trans- Golgi network (TGN46) compartments (Fig. 3A). Therefore the galactosyltransferase-CFP fluorescent pattern allowed us to examine the fate of most Golgi membranes after treatment with siRNA. In contrast to the normal Golgi ribbon seen in cells transfected with a control siRNA, the Golgi complex of cells depleted of RII␣by transfection with the appropriate siRNA appeared fragmented into multiple discrete elements (Fig. 3A). For the most part these fragments remained clustered in the perinuclear region of the cytoplasm. Fragmentation affected all Golgi compartments and occurred in >90% of RII␣-depleted cells whereas a similar effect was observed in <3% of nondepleted cells. Quantitative image analysis indicated that although total Golgi fluorescence was comparable in RII␣- depleted and non-depleted cells, the average number of Golgi fragments per cell increased fivefold in the former (Fig. 3B). Fig. 2. RII␣depletion by siRNA treatment. HeLa cells were incubated (+) or not (–) for 72 hours with an siRNA sequence specific for human RII␣. Alternatively, they were incubated with a control siRNA specific for the rodent form of this protein. (A) Cells were fixed and processed for immunofluorescence with antibodies specific for RII␣and C␣. Cells incubated with siRNA specific for human RII␣were outlined in the image corresponding to C␣ immunostaining. Bars, 10 ␮m. (B) Cells were lysed and processed for immunoblotting to detect both RII␣and C␣. ␤⬘-COP was detected to normalize the amount of protein loaded. (C) Cells were homogenized and total microsomal membranes were prepared. Membranes were lysed and processed for immunoblotting to detect C␣. In this case, protein disulfide isomerase (PDI) was used for normalization. Journal of Cell Science 3767 Golgi organization requires PKA activity Staining with an antibody against ␣-tubulin showed that the microtubule network was mostly unaffected in RII␣-depleted cells (not shown). Furthermore, cells were viable. Particularly, no indication of apoptosis could be appreciated by DNA (Hoechst 33342) staining. Cell adhesion, however, seemed to be hindered because many cells became rounded and detached from the substratum during siRNA treatment. The nature of Golgi fragments originated during RII␣ depletion was investigated by electron microscopy. The typical organization of the Golgi complex in interphase cells consisting of stacks of flattened, elongated cisternae and associated vesicles was replaced in RII␣-depleted cells by an array of globular membranous structures (Fig. 4A). Most of them were found concentrated in the perinuclear region of the cell but apparently they were not connected to each other. Each of these structures consisted of tightly packed curved and partly swollen cisternae and associated vesicles (Fig. 4B). Quite often they show a whorl appearance, with different cisternae arranged concentrically around a centre occupied by vesicles and tubulovesicular structures (Fig. 4C,D). Some of these membranes were found to be positive for HPA-HRP staining indicating that they were derived from the cis-Golgi compartment (not shown). By contrast, the structural organization of other organelles such as the ER, mitochondria, and nuclear envelope was not affected extensively by RII␣ depletion (Fig. 4A). Polarization of Golgi fragments, on the other hand, persisted after disassembly. Thus, not only were protein markers from the different Golgi compartments present in the fragments but they also remained relatively segregated. For instance, the degree of overlapping between cis-Golgi (giantin) and TGN (TGN46) markers seen in the Golgi fragments of RII␣- depleted cells was comparable to that of the intact organelle in control, untreated cells (Fig. 5). Indeed, most fragments showed both cis and trans sides with little or no overlapping (Fig. 5, inset). Additionally, Golgi fragments remained functional in terms of protein transport to the plasma membrane. Thus, the intracellular transport of an anterograde cargo such as the G glycoprotein of the envelope of the vesicular stomatitis virus (VSV-G) was not affected. In particular, this protein reached the plasma membrane with a similar kinetics in both control and RII␣-depleted cells (supplementary material Fig. S1). Effect of PKA displacement on Golgi organization In principle, Golgi disassembly would be triggered by either the absence of RII␣or C␣redistribution. To distinguish between these two possibilities we analyzed the effect of expressing a soluble protein designed to bind RII␣with high affinity. We expected such a protein would trap RII␣and cause redistribution of the complete PKA holoenzyme from the Golgi to cytosol. In eukaryotic cells, PKA targeting to different subcellular structures including the Golgi complex occurs throughout interaction of the RII regulatory subunits with A- kinase anchoring proteins (AKAPs) (Wong and Scott, 2004). Ht31 is a peptide sequence that contains the RII-binding motif of AKAP-Lbc and has been previously used as an effective antagonist of any AKAP-RII interaction (Carr et al., 1992; Rosenmund et al., 1994). As a probe, we used a fusion protein formed by the Ht31 sequence fused to GFP (Alto et al., 2003). Fig. 3. Golgi disassembly induced by RII␣depletion. HeLa cells stably expressing galactosyltransferase-CFP (GalTfase) were incubated for 80 hours with siRNA designed to inhibit the synthesis of either the human or the rodent form of RII␣. (A) Cells were fixed and processed for indirect immunofluorescence with antibodies against the indicated Golgi proteins. Bars, 10 ␮m. (B) Golgiassociated fluorescence and the average number of Golgi fragments per cell was determined from cells (n=12) expressing GalTfase-CFP and incubated or not (–) with either a control siRNA against the rodent form of RII␣or, alternatively, an siRNA against the human version of this protein. Journal of Cell Science 3768 As anticipated, cells expressing this construct showed most PKA redistributed to the cytosol (Fig. 6A). However, depending on the level of expression of Ht31-GFP, the Golgi complex was more or less deprived of RII␣. Yet this organelle underwent collapse and appeared fragmented in most (72%, n=87) cells. As in the case of RII-depleted cells, Golgi fragments (average 33 per cell, Fig. 6B) remained clustered in the perinuclear area and contained protein markers from all the Golgi compartments. It was previously reported that interaction of Ht31 with RII␣could be abolished by preventing amphipathic helix formation, which can be achieved by proline substitution of a crucial isoleucine residue (Hausken et al., 1994). Therefore, as a control, we used the corresponding mutated form of the GFP fusion protein (designated Ht31P- Journal of Cell Science 119 (18) GFP) in our experiments. Irrespective of the level of expression of this construct, RII␣remained associated with the Golgi complex. Furthermore, no change in Golgi structural Fig. 4. Ultrastructural changes in Golgi organization during RII␣depletion. HeLa cells were plated on a dish containing a single coverslip and incubated for 80 hours with siRNA specific for human RII␣. The efficiency of depletion in the culture was first evaluated by immunofluorescence detection of RII␣in cells attached to the coverslip. The rest of the cells were then fixed and processed for transmission electron microscopy. (A) General view of the cytoplasm of RII␣- depleted cells. (B-D) Different Golgi stacks are shown. They appear as whorls composed of multiple concentrically arrayed cisternae. Cisternae appear dilated and fragmented. Bars, 0.5 ␮m. Fig. 5. Assessment of Golgi polarization after disassembly. Non-transfected HeLa cells were incubated (+) or not (–) for 80 hours with siRNA designed to inhibit the synthesis of RII␣. They were fixed and processed for indirect immunofluorescence with antibodies against the indicated endogenous Golgi proteins: giantin to label the cis-Golgi compartment and the TGN marker TGN46. Areas of colocalization (<10% total pixels) are white in the colocalization image. Bar, 10 ␮m. Journal of Cell Science 3769 Golgi organization requires PKA activity organization was noted in cells overexpressing it (Fig. 6A). In summary, these results indicated that Golgi breakdown is the consequence of PKA catalytic activity redistribution rather than RII␣depletion. Golgi disassembly induced by PKA inhibition We next tested whether PKA inhibition would induce a similar effect to that caused by enzyme redistribution. We incubated cells with inhibitors of PKA catalytic activity and looked at the structure of the Golgi complex by immunofluorescence. As shown in Fig. 7A, treatment with either H89 or myristoyl-PKI induced Golgi disassembly. The former is a broad-range kinase inhibitor although at low dose it shows selectivity towards PKA. By contrast, PKI is a potent and specific peptide inhibitor of PKA catalytic subunit. Cells incubated with a low (5-20 ␮M) concentration of either inhibitor exhibited moderate Golgi fragmentation corresponding to 17-20 fragments per cell. Extensive Golgi fragmentation (35-47 fragments per cell) took place in cells exposed to an elevated (30 ␮M or higher) concentration of H89 whereas no further increase in Golgi fragmentation was observed with PKI (Fig. 7B). In order to strictly evaluate the role of PKA in the process, both inhibitors Fig. 6. Golgi disruption caused by PKA displacement. (A) Cells stably expressing galactosyltransferase-CFP (GalTfase) were transfected with cDNA encoding either a wild-type Ht31-GFP construct or the mutated form Ht31P-GFP. 72 hours post transfection, they were fixed and processed for immunofluorescence with anti-RII␣antibody. Bar, 10 ␮m. (B) Cells expressing the indicated Ht31-GFP construct were fixed and processed for immunofluorescence with anti-giantin antibody. Golgi-associated fluorescence and the average number (± s.e.m.) of Golgi fragments per cell were determined (n=12). Fig. 7. Golgi disruption caused by treatment with PKA inhibitors. (A) Non-transfected cells were incubated or not (–) with 20 ␮M of either H89 or myristoyl-PKI (mPKI) for 2 hours at 37°C. They were fixed and processed for immunofluorescence with anti-giantin antibody. (B) The average number (± s.e.m.) of Golgi fragments per cell (n=25) was determined from non-transfected cells incubated for 2 hours at 37°C with the indicated concentrations of each inhibitor and stained for immunofluorescence with anti-giantin antibody. (C) Golgi-associated fluorescence was determined from cells (n=20) incubated or not (–) for 2 hours at 37°C with 20 ␮M of each inhibitor. (D) Cells stably expressing GalTfase-CFP were similarly incubated, fixed and processed for immunofluorescence with antigiantin antibody. Bars, 10 ␮m. Journal of Cell Science 3770 were used at 20 ␮M. Under these conditions, the continuous Golgi ribbon typical of control, untreated cells was replaced by a set of clustered fragments in cells incubated with either inhibitor (Fig. 7D). The time course of Golgi disassembly was also analyzed. Whereas the number of Golgi fragments per cell did not vary significantly from 30 minutes to 2 hours of incubation in the presence of PKI, a slight increase from 10 to 19 fragments per cell occurred during the same time period in cells exposed to H89. Golgi-associated fluorescence, on the other hand, did not vary with either inhibitor (Fig. 7C). The structural features of Golgi fragments generated during PKI or H89 incubations resembled those generated during RII␣ depletion by siRNA treatment. In particular, Golgi stacks became globular structures formed by concentrically arrayed, curved, swollen cisternae (Fig. 8). Together, these results indicated that Golgi-associated PKA activity plays a relevant role in the maintenance of the structural organization of this organelle. We concluded that either prolonged inhibition of the Journal of Cell Science 119 (18) PKA catalytic activity or displacement from the Golgi gives rise to disruption of the Golgi ribbon. We next asked whether it would be also involved in the assembly of this structure. Role of PKA activity in Golgi biogenesis We first examined the subcellular localization of PKA subunits under conditions of Golgi redistribution into the ER. For instance, transient expression of dominant-negative mutated forms of the COPII component Sar1p interferes with the function of this coat complex and this blocks protein export from the ER (Kuge et al., 1994; Ward et al., 2001). Since the Golgi-ER recycling pathway persists under these conditions, all the Golgi transmembrane proteins are finally relocated to the ER. The exact point of transport block differs, however, depending on the particular mutated version being expressed. Expression of a GDP-restricted form of Sar1p, designated T39N, prevents the assembly of the COPII complex and this causes disassembly of the ER exit sites (Kuge et al., 1994; Stroud et al., 2003; Ward et al., 2001). By contrast, expression of a GTP-restricted form of Sar1p, designated H79G, inhibits export from the ER exit sites which otherwise assemble normally (Miles et al., 2001; Ward et al., 2001). We found that both PKA subunits were distributed dispersed throughout the cytosol in cells expressing the constitutively inactive T39N form of Sar1p. By contrast, in cells expressing the permanently active H79G mutant form of Sar1p PKA subunits were concentrated at the ER exit sites (Fig. 9A). PKA association with the centrosome remained unaltered in both cases. These findings raised the possibility that PKA activity could regulate protein export from the ER by influencing the assembly and organization of the ER exit sites. To test this, we examined the distribution of sec13, another component of the COPII complex and a marker of the ER exit sites (Tang et al., 1997). No difference in the punctate staining pattern of sec13 was noted in cells depleted of RII␣by siRNA treatment in comparison to control, non-depleted cells (Fig. 9B). A similar result was obtained with cells expressing Ht31-GFP or Fig. 8. Ultrastructural changes in Golgi organization following PKA inhibition. Cells were incubated at 37°C with 20 ␮M of either H89 (A-D) or myristoyl- PKI (E) for 2 hours before fixation and processing for transmission electron microscopy. (F) The Golgi ribbon of a control, untreated cell is shown for comparison. Bars, 0.5 ␮m. Journal of Cell Science 3771 Golgi organization requires PKA activity incubated with PKA inhibitors (not shown). Therefore, the number and distribution of the ER exit sites does not seem to be determined by the localization and activity of PKA. In a different approach, we looked at the role of PKA activity in the Golgi reassembly process during reversal from brefeldin A (BFA) treatment (Fig. 10). In BFA-treated cells, PKA subunits were localized dispersed throughout the cytosol whereas Golgi transmembrane proteins (exemplified in Fig. 10 by the galactosyltransferase-CFP construct) showed a reticular fluorescent pattern characteristic of the ER. During early reversal times (5-10 minutes) Golgi proteins started to emerge from the ER at multiple peripheral sites. Subsequently (20 minutes) they clustered to the perinuclear region of the cell. With time these intermediates coalesced to form a continuous Golgi ribbon, which became evident by 40-60 minutes of recovery. Interestingly, recruitment of the PKA holoenzyme to the Golgi membranes occurred late in the reassembly process. Thus, most of the Golgi fluorescent spots detected at early reversal times did not contain associated PKA (Fig. 10, 10-20 minutes). Indeed, even at late time points, newly emerging Golgi membranes as opposed to the reassembled Golgi ribbon lacked associated PKA (Fig. 10, 40 minutes). These observations suggested that PKA activity might play a role in a final stage of the reassembly process. To analyze this further we studied the effect of PKA inhibition on Golgi reconstruction. Cells expressing galactosyltransferase-CFP were treated with H89 during recovery from BFA incubation (Fig. 11). In the continuous presence of H89 Golgi membranes emerged from the ER at much lower rate than in cells not incubated with this agent. The sequence of events was, however, similar. Initially, Golgi components exited the ER at multiple peripheral sites (Fig. 11A, 30 minutes) and later on (1-2 hours) they became clustered in the juxtanuclear region. The reassembly process seemed to be arrested at this stage because complete reconstruction of the Golgi complex did not occur even after prolonged incubations (Fig. 11A, 3 hours). In particular, no Golgi ribbon was formed in the presence of H89. Instead, a number of disconnected elements remained clustered in the perinuclear region. These were characterized by electron microscopy as whorl-like membranous structures composed of concentrically arrayed cisternae (Fig. 11C). Apparently, they were final intermediates in the reassembly process because the continuity of the Golgi ribbon was readily re-established upon H89 removal (Fig. 11A, 3 hours + 1 hour). These data, therefore, indicated that PKA catalytic activity is required at a final step of the Golgi reassembly process, that is, the formation of a continuous ribbon structure from separate Golgi stacks. PKA requirement, on the other hand, corresponded to the recruitment of the holoenzyme from the cytosol to the Golgi membranes during reassembly. Discussion The functional significance of PKA association with the Golgi complex of mammalian cells is a mystery. Since its subcellular localization was first reported two decades ago (Nigg et al., 1985b) this kinase has been recognized as an outstanding member of the set of signaling and regulatory molecules associated to the Golgi under steady-state conditions. With the exception of a role in the regulation of transport activities (Cabrera et al., 2003; Martín et al., 2000; Muñiz et al., 1996; Muñiz et al., 1997), however, there is no clear indication on the implication of Golgi-associated PKA in any relevant event. In this report, we provide data indicating that Golgi-associated PKA activity plays a relevant role in the assembly and maintenance of the continuous ribbon structure, a characteristic feature of the Golgi organization in mammalian cells. Recently, this ribbon-like architecture was shown to determine the efficiency of glycoprotein processing by allowing even distribution of glycosyltransferases among different Golgi stacks (Puthenveedu et al., 2006). Golgi-associated PKA could therefore contribute to the structural integrity and functional uniformity of this organelle. A structural role for Golgi-associated PKA activity is suggested from data derived from the application of photobleaching techniques to living cells. The analysis of the dynamics of association to and dissociation from the Golgi of RII␣indicated that this protein is stably associated with the cytosolic surface of Golgi membranes and, in contrast to other Golgi peripheral proteins, does not exchange rapidly with the population of soluble molecules. Indeed, a significant fraction of Golgi-associated RII␣did not exchange at all and behaved Fig. 9. Role of PKA in the organization of ER exit sites. (A) Presence of PKA at the ER exit sites. Cells overexpressing CFP- tagged forms of the indicated mutated versions of Sar1p were fixed and processed for immunofluorescence with antibodies to C␣. (B) PKA displacement does not affect the distribution of ER exit sites. Cells incubated for 80 hours with siRNA to target either the human or the rodent form of RII␣were fixed and processed for immunofluorescence with antibodies to Sec13p. Staining of the centrosome is indicated with arrowheads. Bars, 10 ␮m. Journal of Cell Science 3772 as immobile molecules. To reveal such a structural role, three experimental strategies were used. First, cells were depleted of RII␣by siRNA treatment. PKA targeting to different subcellular structures including the Golgi complex is achieved through association of the regulatory RII subunits to AKAPs (Michel and Scott, 2002; Wong and Scott, 2004). The catalytic C subunits, in turn, are RII bound. Accordingly, RII␣depletion by siRNA treatment gave rise to C␣redistribution from the Golgi complex to the cytosol. Second, cells were transfected with a plasmid encoding Ht31-GFP. This construct interferes with the association of RII␣to AKAPs and in our study caused cytosol redistribution of the complete PKA holoenzyme allowing us to evaluate the consequences of PKA displacement without affecting enzyme expression. Third, and most significantly, cells were incubated with either H89 or myristoyl-PKI to inhibit PKA catalytic activity. Results were similar in all three cases and showed that the structural organization of the Golgi complex depends on PKA association. Thus, following PKA pharmacological inhibition Journal of Cell Science 119 (18) or its displacement from the Golgi, this organelle appeared disrupted into a number of fragments that remained clustered in the perinuclear area of the cell. We conclude that the integrity of the Golgi ribbon requires both proper location and enzymatic activity of the PKA holoenzyme. Golgi fragments were polarized as judged by the distribution of both cis and trans (TGN) markers. This suggests that the mechanism responsible for Golgi disassembly might involve unlinking of the continuous Golgi ribbon into separate stacks. For instance, PKA phosphorylation of a structural protein could be required in order to maintain Golgi ribbon organization. Golgi unlinking would then occur whenever this modification is somehow abrogated. Alternatively, the Golgi complex could undergo fragmentation as the result of a block in ongoing protein trafficking. This, however, does not seem to be the case because Golgi fragments maintain at least their competence to transport VSV- G to the plasma membrane. Also, accumulation of transport vesicles was not observed in either RII␣- depleted cells or cells treated with PKA inhibitors. The effect caused by either PKA inhibition or mislocalization on Golgi structure would therefore be similar to that caused by GM130 knockdown (Puthenveedu et al., 2006). Further studies will be needed to characterize the Golgi proteins that are substrates of PKA activity and to determine how this modification affects Golgi organization. The involvement of PKA activity in Golgi biogenesis was established by studying the organelle reassembly during recovery from BFA treatment. PKA subunits were recruited from the cytosol to the Golgi membranes as these emerged from the ER and fused to form a readily identifiable Golgi ribbon. The time of association, however, was clearly delayed in comparison to the time of re-emergence from the ER. In agreement with previous studies (Bannykh et al., 2005; Puri and Linstedt, 2003), Golgi membranes exited the ER at multiple peripheral sites that with time converged to the juxtanuclear region where they fused and reconstructed the ribbon organization. PKA association was coincident with ribbon formation. Thus, most Golgi intermediates detected at early times of the reassembly process lacked associated PKA. Furthermore, PKA inhibition during reversal from BFA treatment blocked ribbon formation by preventing the fusion of Golgi elements previously clustered in the perinuclear area. These elements corresponded to globular Golgi stacks composed of multiple concentrically arranged cisternae. Interestingly, similar structures have been recently reported to occur in cells recovering from BFA treatment in the absence of any other additional agent (Bannykh et al., 2005). This suggests that they might represent transient intermediates in the normal process of Golgi reassembly from the ER. Golgi fragments generated upon PKA displacement or inhibition also showed similar morphology. Therefore, the available evidence indicates that PKA activity is involved in the assembly and maintenance of the ribbon architecture typical of the mammalian Golgi complex. Fig. 10. PKA recruitment during Golgi reconstruction. Cells stably expressing GalTfase-CFP were incubated with 2 ␮g/ml BFA for 1 hour at 37°C and then allowed to recover for the indicated time periods. They were fixed and processed for immunofluorescence with anti-RII␣antibody. Newly formed Golgi fragments containing (arrowheads) or not (arrows) associated RII␣are indicated. Bar, 10 ␮m. Journal of Cell Science