Endosome-to-Golgi Transport Is Regulated by Protein Kinase A Type IIα*

Studies of RIIα-deficient B lymphoid cells and stable transfectants expressing the type IIα regulatory subunit (RIIα) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type IIα mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RIIα-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RIIα in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type IIα seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RIIα expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RIIα were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type IIα isozyme.

The mechanism by which different extracellular ligands that mediate their signals through the same second messenger might give rise to a specific intracellular response has been the subject of intensive research for several years (1). In the case of cAMP signal transduction, it has been demonstrated that the subcellular localization of protein kinase A (PKA) 1 is important for the specificity (1).
PKA is composed of two catalytic (C) subunits and one reg-ulatory dimer (R 2 ) that in the absence of cAMP form an inactive heterotetramer (R 2 C 2 ). Upon binding of cAMP to the R subunits, the enzyme dissociates and releases two free, active C subunits (2). The R 2 dimer is also implicated in the targeting of different PKA isoforms to various intracellular locations and to specific substrates through interactions with protein kinase A-anchoring proteins (3). Four different isoforms of the R subunit have been identified as products of separate genes in mammalian cells, and they have been termed RI␣, RI␤, RII␣, and RII␤ (1,2,4). They all contain two C-terminal cAMP binding sites, a hinge region that interacts with and inhibits the catalytic subunit, and a dimerization domain responsible for the interaction between the two regulatory subunits that make up the regulatory dimer of PKA (2). Whereas RI subunits are known to be mainly soluble, RII subunits are primarily associated with cytoskeletal elements and membranes (1,3). Studies of several human cell lines have revealed that the PKA type II␣ isozyme (containing RII␣ and C␣) is concentrated in centrosomes and in Golgi-associated compartments (5,6). In contrast, PKA type II␤ (containing RII␤) is associated more selectively with the centrosomal region and not with Golgi structures (5). Based on the important role of the Golgi apparatus in intracellular transport and protein sorting and the localization of PKA, which previously has been implicated in vesicle-mediated protein transport processes (7)(8)(9)(10), the possibility existed that a distinct Golgi-associated pool of PKA type II␣ isozyme was involved in regulation of transport through this organelle. To investigate whether PKA type II␣ is involved in the regulation of retrograde transport, we studied the transport of the plant toxin ricin.
Ricin belongs to a family of plant and bacterial toxins that enter cells via the endocytic pathway. The toxin is transported retrogradely through the Golgi to the endoplasmic reticulum (ER) before it enters the cytosol, where it inhibits protein synthesis (11,12). Ricin consists of an A-chain and a B-chain that are linked by a disulfide bridge. The B-chain binds to terminal galactose in both glycolipids and glycoproteins at the plasma membrane, whereas the A-chain enzymatically inhibits the protein synthesis after entry into the cytosol (12). Because ricin binds to both glycolipids and glycoproteins at the plasma membrane, it will be endocytosed by any vesicle that pinches off. Once ricin is endocytosed, it can be transported through the endosomal compartments, recycled back to the plasma membrane, delivered to the lysosomes, or transported retrogradely to the TGN and to the ER (11,12).
In this study, we took advantage of a RII␣-deficient B lymphoid cell line, Reh (13), and reintroduced RII␣ by making stable transfectants with a Golgi-associated pool of PKA type II␣ to examine the effect on intracellular transport. We used two different ricin constructs to monitor the retrograde toxin trans-port. Within the Golgi apparatus, recombinant ricin with a tyrosine sulfation site (ricin sulf-1) becomes radiolabeled in the presence of radioactive sulfate (14). This has made ricin sulf-1 a valuable tool to study intracellular transport to the TGN. A recombinant ricin construct with a sulfation site and three overlapping N-glycosylation sites (ricin sulf-2) is both modified in the TGN and N-glycosylated in the ER (14). The N-glycosylation of the toxin results in a molecular shift that can be observed on SDS-PAGE. This construct has therefore been used to study intracellular transport to the ER. As shown in the present study, the expression of PKA type II␣ (RII␣) on a negative background in a lymphoid cell line leads to modulation of the retrograde transport of ricin, indicating a regulatory role for Golgi-associated PKA type II␣ on these transport steps.
Cells and Cell Culture-A human B-lymphoid cell line (Reh) stably transfected with pMep4 vector (clone pMep) or RII␣ under direction of the human metallothionein IIA promoter (clone RII␣) (15) was maintained under standard conditions (5% CO 2 in RPMI 1640 medium containing 5% (v/v) FCS, 2 mM L-glutamine, and 100 g/ml streptomycin). Every third month, the cells expressing RII␣ were incubated under standard conditions in the presence of 200 g/ml hygromycin B. On the day that the experiments were performed, the cells were seeded into Eppendorf tubes at a density of 8 ϫ 10 5 cells/tube.
Measurement of Protein Synthesis-The cells were washed twice with HEPES medium (bicarbonate-free Eagle's minimum essential medium buffered with 20 mM HEPES to pH 7.4), and then incubated with the same medium for 30 min at 37°C. The samples were then incubated in the presence or absence of 350 M 8CPT-cAMP for 30 min before 1, 10, and 100 ng/ml of ricin were added to the cells, which were further incubated for 30 min at 37°C. The cells were incubated thereafter with HEPES medium containing 1 Ci/ml [ 3 H]leucine for 20 min at 37°C, extracted with 5% (w/v) trichloroacetic acid for 10 min followed by a brief wash with the same solution. Subsequently, cells were dissolved in 0.1 M KOH, and the acid-precipitable radioactivity was measured. The results are presented as percentage of radioactivity incorporated in cells incubated without toxin. The concentration of ricin required to inhibit the protein synthesis by 50% was chosen as a measure of the sensitivity of cells to ricin. Variation between duplicate measurements was less than 15%.
Sulfation of Ricin Sulf-1 and Sulf-2-Recombinant ricin A-sulf-1 and ricin A-sulf-2, modified to contain a tyrosine sulfation site and both a tyrosine sulfation site and three overlapping N-glycosylation sites, respectively, were expressed, purified, and reconstituted with ricin Bchain (ricin sulf-1 and ricin sulf-2, respectively) according to the procedure described previously (14). The cells were washed twice in sulfatefree RPMI 1640 medium that contained 2 mM L-glutamine, and then incubated with 0.1 mCi/ml Na 2 35 SO 4 in the same medium for 3 h. The cells were then incubated in the presence or absence of 350 M 8CPT-cAMP and/or 20 g/ml cycloheximide for 30 min at 37°C, before ricin sulf-1 or ricin sulf-2 (ϳ300 ng/ml) was added. The incubation was continued for 2 h at 37°C. The cells were then washed twice for 5 min at 37°C with HEPES medium that contained 0.1 M lactose followed by cold PBS (140 mM NaCl and 10 mM Na 2 HPO 4 , pH 7.2). The cells were thereafter lysed (lysis buffer, 0.1 M NaCl, 10 mM Na 2 HPO 4 , 1 mM EDTA, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mM aprotinin, pH 7.4), and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was immunoprecipitated overnight at 4°C with rabbit anti-ricin antibodies immobilized on protein A-Sepharose. The beads were then washed twice with PBS containing 0.35% (v/v) Triton X-100, and the immunoprecipitated material was analyzed by SDS-PAGE (12%) under reducing conditions. SDS-PAGE-SDS-PAGE was carried out in the presence of ␤-mercaptoethanol as described previously (16). The gels were fixed in 4% acetic acid (v/v) and 27% (v/v) methanol for 30 min and then incubated with 1 M sodium salicylate, pH 5.8, in 2% (v/v) glycerol for 30 min. The dried gels were then exposed to Kodak XAR-5 films (Eastman Kodak Co.) at Ϫ80°C for autoradiography.
Immunofluorescence Microscopy-For analysis of ricin distribution, ricin was labeled with Cy5 (Amersham Biosciences) according to the manufacturer's instructions. The coverslips were coated with poly(Dlysine) (M r ϭ 150 000) as described previously (17). The cells were washed twice with HEPES medium before addition of Cy5-labeled ricin (ϳ1000 ng/ml). After incubating the cells for 30 min at 37°C, they were washed with cold PBS and further incubated with 3% (w/v) paraformaldehyde in PBS for 15 min at room temperature. The cells were then washed 3 times with PBS before incubation with 0.1% (v/v) Triton X-100 dissolved in PBS for 5 min at room temperature. Subsequently, the cells were washed in PBS, and incubated with PBS containing 5% (v/v) FCS for 30 min. The permeabilized cells were incubated with rabbit anti-human RII␣ (1:1000) or with mouse mAb CTR 433 (1:10) to label the medial Golgi compartment, with mouse mAb GM130 (1:1000) to label the cis Golgi, or with sheep anti-human TGN46 (1:100) to label the trans Golgi in PBS containing 5% (v/v) FCS for 30 min at room temperature. The cells were then washed three times for 5 min with PBS containing 5% (v/v) FCS followed by incubation with FITC-labeled goat anti-mouse antibody (1:100) to detect CTR 433 and GM130, with Cy3-labeled goat anti-rabbit antibody (1:500) to detect RII␣, or with FITC-labeled donkey anti-sheep/goat antibody (1:100) to detect TGN46 in PBS containing 5% (v/v) FCS. After staining, the cells were washed three times for 5 min with PBS at room temperature, and the coverslips were mounted in Mowiol. Immunofluorescence microscopy was performed using a Leica (Wetzlar, Germany) confocal microscope. Images were captured with a resolution of 1024 ϫ 768 pixels and prepared with the use of Adobe Photoshop 4.0 (Adobe Systems, Mountain View, CA).
For analysis of PKA distribution versus centrosomal marker, cells were fixed in 4% paraformaldehyde in PBS for 20 min at 37°C, rinsed twice in PBS, and incubated for 10 min with 50 mM ammonium chloride in PBS. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS with 0.2% BSA. Primary antibodies were diluted in PBS containing 3% BSA to concentrations of 140 ng/ml for mAb CTR 453 (a centrosomal marker), 100 ng/ml for rabbit anti-human RII␣, 500 ng/ml for rabbit anti-human RII␤, and 1 g/ml for mouse anti-RII␣ mAb and incubated for 1 h at room temperature. Cells were then washed three times in PBST (PBS with 1% Tween 20) to remove unbound antibodies followed by incubation with fluorochrome-conjugated secondary antibodies (FITC and Texas Red) for 1 h at room temperature. Finally, the cells were mounted in CITIFLUOR (Citifluor, London, UK). Confocal microscopy was performed on a Sarastro 2000 confocal microscope (Amersham Biosciences), equipped with an argon laser (488 to 514 nm wavelength). Ten sections of 0.25 m (averaging five full frames of the same section) were scanned, and stacks of optical sections for each data set were compiled with Voxel View software on an IRIS 4D-70 GT graphics work station (SGI, Mountain View, CA).
Intracellular Accumulation of Ricin-Ricin was 125 I-labeled according to the procedure described by Fraker and Speck (18) to a specific activity of 5 ϫ 10 4 cpm/ng. The intracellular accumulation of 125 Ilabeled ricin was measured after 2 h at 37°C as the amount of toxin that could not be removed by lactose treatment, as described previously (19). The cells were preincubated with 350 M 8CPT-cAMP for 30 min at 37°C.
Ricin Endocytosis-The endocytosis of ricin was measured using the ORIGEN analyzer (IGEN Inc., Rockville, MD). Ricin was labeled with N-hydroxysuccinimide ester-activated tris(bipyridine) chelated ruthenium(II) TAG (IGEN Inc.) according to the manufacturer's instructions and simultaneously biotinylated with reducible immunopure NHS-SS-Biotin (Pierce). The cells were washed with HEPES medium and then incubated in the presence or absence of 350 M 8CPT-cAMP for 30 min at 37°C, followed by addition of TAG-labeled ricin (25 ng/ml) to allow endocytic uptake of the toxin for 30 min at 37°C. Half of the samples were then treated with 0.1 M MESNA for 1 h on ice (20), and the other half of the samples was washed in cold PBS. The cells were then lysed (lysis buffer, 100 mM NaCl, 5 mM MgCl 2 , 50 mM HEPES, and 1% (v/v) Triton X-100) for 10 min on ice. Ricin that is both TAG-labeled and biotinylated can be detected in the lysate by using streptavidin-conjugated beads (Dynal, Oslo, Norway) and the ORIGEN analyzer (IGEN Inc.). The endocytosis of TAG-labeled ricin was measured as the amount of toxin that could not be removed by MESNA treatment as described previously (21).
Miscellaneous-Detection of PKA subunits by Northern blot analysis or [ 32 P]8-azido-cAMP photoaffinity labeling and immunoprecipitation was performed essentially as described previously (13).

Expression of PKA-RII␣ in a RII␣-deficient, B Lymphoid Cell
Line, Reh-To study effects related specifically to expression of RII␣, stably transfected cell lines expressing RII␣ under control of the zinc-inducible type IIa metallothionein promoter were made together with control clones transfected with empty plasmid by selection on hygromycin B. Fig. 1 shows characterization of one clone expressing RII␣ compared with a control clone. The RII␣ transfected clone displayed high levels of RII␣ mRNA (A) as well as a cAMP-binding protein, immunoreactive with RII␣ antibodies and present in both the soluble S200 and detergent-soluble Tx-100 fractions (B). In contrast, a control-transfected clone did not have any detectable RII␣ mRNA or protein. Both cell clones had equal levels of RII␤ mRNA. We conclude that the RII␣-transfected clone expresses RII␣ at quite high levels even in the absence of zinc.
PKA-RII␣ Expressed in Reh Cells Is Targeted to the Golgi-Centrosomal Region-It has previously been shown that RII␣ is localized to the Golgi-centrosomal area in SaOS2 osteosarcoma cells and in COS-7 cells (5, 6). We have examined, by immunofluorescence and confocal microscopy, whether this was also the case when RII␣ was expressed in Reh cells. Double staining of the centrosomal marker CTR 453 (22) (Figs. 2, A, C, and E) and RII␣ (Figs. 2, B, D, and F) demonstrated that RII␣ was absent in wild-type cells (Fig. 2B), but appeared in the centrosomal region when expressed (Fig. 2, D and F versus C  and E). Furthermore, the distribution of RII␣ was wider than that of the centrosomal protein CTR 453 (Fig. 2, F versus E). Because RII␤ is targeted to centrosomes and present in Reh cells, as in most cancer cell lines [ Fig. 1; Ref. 5), we next examined the distribution of RII␣ versus that of RII␤ by dual staining and image overlays (Fig. 2G). Again, RII␣ (red) was found in a wider area than RII␤ (green, overlap seems yellow), which in separate experiments showed a distribution overlapping well with that of the centrosomal marker, CTR 453 (not shown). In addition, we performed immunofluorescence studies of the localization of RII␣ and the Golgi markers GM130 and TGN46. As shown in Fig. 3, RII␣ was partially colocalized with both Golgi markers. Together, these experiments demonstrated that clones expressing RII␣ acquired a new PKA isozyme that localizes in the centrosomal-Golgi region. To explore the function of this particular pool of Golgi-associated Total RNA was extracted in guanidinium isothiocyanate and prepared by CsCl-gradient centrifugation, subjected to electrophoresis, and blotted onto nylon membrane. The resulting filter was hybridized with radiolabeled cDNA probes for RII␣ and RII␤. B, stably transfected clones were lysed in isotonic sucrose buffer by ultra Turrax homogenization, nuclei were pelleted at 500 ϫ g, the postnuclear supernatant was subjected to a 200,000 ϫ g spin for 1 h to yield a soluble fraction (S200), and a pellet was solubilized in isotonic sucrose buffer with 0.5% Triton X-100 and centrifuged at 15,000 ϫ g to remove insoluble material (Tx-100). The soluble S200 and detergent-soluble Tx-100 fractions were next subjected to photoaffinity labeling with [ 32 P]8-azido-cAMP in the presence (ϩ) or absence (Ϫ) of excess cold cAMP to compete binding. Ten percent of the samples were then boiled directly in SDS-sample buffer (lanes 1, 2, 5, and 6), whereas the rest were used for immunoprecipitation with antibodies to RI␣ and RII␣ before analysis by SDS-PAGE and autoradiography (lanes 3, 4, 7, and 8). Arrows indicate RI␣ (top and bottom) and RII␣ (top). B) and a clone stably expressing RII␣ (C to F) were double stained with a centrosomal marker (mAb CTR 453; A, C, and E) and hRII␣ antibodies (B, D, and F), and confocal images from sections in the centrosomal area are shown separately in pseudo-color, ranging from no signal (blue) to high signal (yellow to red). G, confocal image after double staining with mAb RII␣ (red) and hRII␤ Abs (green). Bars: A to D, 5 m; E to G, 2 m. Arrows indicate centrosomes. PKA, we next examined the intracellular transport of ricin in Reh cells in the presence and absence of a Golgi-associated pool of PKA type II␣.

FIG. 2. RII␣ is targeted to the Golgi-centrosomal region when expressed in an RII␣-deficient, B lymphoid cell line, Reh, and concentrates in a wider area than RII␤. Human wild-type Reh cells (A and
Intoxication of Lymphocytes with Ricin Is Affected by RII␣ Expression-To investigate whether one or more steps along the intracellular route followed by ricin are affected by RII␣, we tested the sensitivity to ricin of clone RII␣ cells with and without the stimulation of PKA and compared it with that of cells deficient in PKA type II␣ (clone pMep). To activate PKA, we used a cell-permeable cAMP analog, 8CPT-cAMP, with high affinity for the type II regulatory subunits (23,24). As shown in Fig. 4, cells expressing RII␣ were ϳ2-fold more sensitive to ricin than the RII␣-deficient cells, and although addition of 8CPT-cAMP had a sensitizing effect on the pMep cells, it sensitized the cells expressing RII␣ to a much larger extent. Similar experiments with untransfected cells (Reh), as well as with other clones transfected with RII␣ with similar targeting, showed the same pattern of sensitivity (results not shown). Thus, RII␣ regulates one or several steps on the route of ricin to the cytosol in lymphoid cells, although RII␣ is not strictly required for ricin intoxication.
Sulfation of Ricin Sulf-1 in Cells with or without RII␣-To study the transport of ricin from the plasma membrane to the Golgi apparatus, recombinant ricin sulf-1 that contains a tyrosine sulfation site was used. The cells were incubated in the presence or absence of 8CPT-cAMP and ricin sulf-1 (Fig. 5). Because the protein synthesis was somewhat stimulated in cells preincubated with 8CPT-cAMP, and an increased transport of newly synthesized proteins through the TGN could in theory result in an increased competition for sulfation and thus interfere with the assay; some cells were also incubated with cycloheximide to inhibit protein synthesis. Fig. 5C shows that the sulfation of ricin in control cells increased by ϳ70% in the presence of 8CPT-cAMP, whereas the sulfation increased by ϳ120% in the cells expressing RII␣. However, in cells treated with both 8CPT-cAMP and cycloheximide, the sulfation of ricin increased by ϳ80% in the control cells and by ϳ250% in the RII␣-expressing cells. This result indicates that when PKA is activated, the transport of ricin to the Golgi apparatus is increased to a larger extent in cells expressing RII␣ than in control cells.
Sulfation and Glycosylation of Ricin Sulf-2 in Cells with and without RII␣-To further investigate the transport of ricin to the ER, recombinant ricin sulf-2 that contained a tyrosine sulfation site and three overlapping N-glycosylation sites was used. When ricin sulf-2 was added to control cells (clone pMep) or to RII␣-expressing cells (clone RII␣) in the presence of ra-dioactive sulfate and immunoprecipitated from cell lysates, two bands were visible (Figs. 6, A and B). The upper molecular weight band represents ricin that has been both sulfated and glycosylated, and the lower molecular weight band represents ricin that has only been sulfated. As shown in Fig. 6C, the amount of ricin in the ER, measured as sulfated and glycosylated ricin relative to the total amount of sulfated ricin, was significantly increased in cells expressing RII␣ (clone RII␣) compared with the control cells (clone pMep) when PKA was activated by 8CPT-cAMP. These observations indicate that not only the transport of ricin to the Golgi apparatus but also the further transport of the toxin to the ER is increased by RII␣ in the presence of 8CPT-cAMP.
Intracellular Accumulation of Ricin in Cells with and without RII␣-The increased sulfation of ricin sulf-1 and ricin sulf-2 observed in cells expressing RII␣ compared with control cells deficient in RII␣ could be caused by an increased binding and endocytosis of ricin or by an increased endosome-to-Golgi transport. We therefore investigated the endocytosis of ricin in the presence or absence of 8CPT-cAMP. Fig. 7 demonstrates that the accumulation of ricin after 2 h of incubation was not significantly changed by addition of 8CPT-cAMP or by the expression of RII␣. Similar data were obtained when the cells were incubated with ricin for 30 min (results not shown). In addition, the binding of ricin to the plasma membrane was not significantly altered by 8CPT-cAMP or by expression of RII␣ (data not shown). Thus, the increased transport of ricin to the Golgi apparatus cannot be accounted for by a change in the endocytosis of ricin.
Colocalization of Ricin and RII␣ in the Golgi Area-As evident from Figs. 2 and 3 and previous reports (5), RII␣ mainly exhibits a perinuclear, Golgi-associated localization that is detergent-extractable, indicating membrane-associated localization. To investigate whether RII␣ is associated with ricincontaining structures, immunofluorescence studies were performed using antibodies raised against human RII␣ (Fig.  8A, visualized by Cy3-labeled secondary antibodies, red), Cy5labeled ricin (Fig. 8B, blue), and the medial Golgi marker CTR 433 (Fig. 8C, visualized by FITC-labeled secondary antibodies, green). Both RII␣ and ricin seemed colocalized (Fig. 8F) and were also found to partly colocalize with the medial Golgi marker (Fig. 8, D and E, respectively).
It has been reported that both the type II␣ and II␤ regulatory subunits are associated with centrosomes (5). Because the previous experiment showed colocalization between ricin and RII␣, we investigated the distribution and localization of ricin in the centrosomal area. We showed that whereas minor amounts of RII␣ are present in centrosomes, no ricin was detected in this region (data not shown).

DISCUSSION
In the present study, we have investigated the influence of the Golgi-associated type II␣ regulatory subunit of PKA on the intracellular transport of ricin. RII␣ was expressed on a negative background, and 8CPT-cAMP was used to activate PKA. Because phenotypic differences between clones may arise (e.g. relating to incorporation in the genomic DNA), we investigated several clones expressing RII␣ with similar results. Different clones transfected with empty vector displayed a phenotype similar to that of wild-type cells.
The first indication that RII␣ might be involved in regulation of intracellular transport was found by investigating the entry of the plant toxin ricin into the cytosol, measured as ricin toxicity. Even when PKA was not activated by addition of Cells were washed in sulfate-free RPMI 1640 medium and incubated with 0.1 mCi/ml Na 2 35 SO 4 for 3 h, and then further incubated in the presence or absence of 8CPT-cAMP and/or cycloheximide for 30 min at 37°C before ricin sulf-1 was added. The incubation was continued for 2 h at 37°C before the cells were washed with lactose and cold PBS before cell lysis, as described under "Experimental Procedures." Nuclei were removed by centrifugation, and ricin was immunoprecipitated overnight at 4°C with rabbit anti-ricin antibodies immobilized on protein A-Sepharose. The immunoprecipitated material was analyzed by SDS-PAGE (12%) under reducing conditions followed by autoradiography. C, levels of ricin sulfation in the presence of 8CPT-cAMP are calculated relative to levels in untreated cells, whereas sulfation levels in cells treated with both cycloheximide and 8CPT-cAMP are calculated relative to cells incubated with cycloheximide alone by densitometric scanning of bands. Error bars show half range between duplicates of lanes from the experiment shown in A and B. The intensities of the resulting bands were determined by densitometric quantitation using ImageQuant 5.5 (Amersham Biosciences).

FIG. 6. The effect of 8CPT-cAMP on the sulfation and glycosylation of recombinant ricin A-chain with sulfation and glycosylation sites (ricin A-sulf-2) in cells deficient in (clone pMep) or expressing RII␣ (clone RII␣).
A and B, autoradiograms from representative experiments with ricin sulfation and N-glycosylation in pMep (A) and RII␣ cells (B), respectively. The higher molecular weight bands represent ricin A-sulf-2 that has been both sulfated and glycosylated, whereas the lower molecular weight bands represent ricin A-sulf-2 that has been sulfated only. C, the intensities of the upper molecular weight bands were calculated relative to the sum of both the upper and lower molecular weight bands for the different conditions listed. The cells were treated as described in the legend to Fig. 5. external 8CPT-cAMP, the cells expressing RII␣ were about 2-fold more sensitive to ricin than the control cells. The most likely explanation seems to be that there is a certain level of endogenous cAMP that partly activates PKA. This explanation was strengthened by the finding that the transfected cells were about 4-fold more sensitive to ricin than the control cells when PKA was activated by addition of external 8CPT-cAMP. Also, the control cells were shown to be slightly more sensitive to ricin when PKA was activated. This might be caused by the activation of other isozymes of PKA that also regulate intracellular transport but, apparently, less efficiently than PKA type II␣. The PKA type II␣ might be a better regulator than the other PKA isozymes because it is closer to the vesicular route of ricin transport into the Golgi apparatus. It might be a question of the local concentration of PKA whether it serves as a good regulator.
It has previously been shown that addition of 8-bromo-cAMP to Madin-Darby canine kidney cells gives a selective stimulation of the transport of apically internalized ricin to the Golgi apparatus (25) (Fig. 9). However, in those cells, we cannot ascribe this regulation to a Golgi-located PKA. We here demonstrate that the sulfation of ricin is increased in cells expressing RII␣ compared with control cells when PKA was activated by externally added 8CPT-cAMP. This result indicates that RII␣ is a strong regulator of the transport of ricin from the plasma membrane to the Golgi apparatus. Earlier studies have demonstrated that calmodulin (26) and calcium (27) can modulate the transport of ricin from endosomes to the Golgi apparatus in other cell lines (Fig. 9). Clearly, different factors are involved in the regulation of retrograde transport.
Interestingly, confocal microscopy demonstrated a localization of both ricin and RII␣ in the Golgi area. The difference in ricin sulfation was much larger when both cell types were preincubated in the presence of cycloheximide. Such experiments were performed because the protein synthesis was somewhat stimulated in cells incubated with 8CPT-cAMP (data not shown), and an increased transport of newly synthesized proteins through the TGN in theory could result in an increased competition for sulfation. Even though it cannot be excluded that cycloheximide might result in an increased transport of ricin to the Golgi apparatus, the increased sulfation of ricin that was observed in cells expressing RII␣ compared with the control cells in the presence of 8CPT-cAMP and cycloheximide strongly supports the notion of a regulatory role of RII␣ in intracellular transport from the plasma membrane to the Golgi apparatus.
The increased transport of ricin to the Golgi apparatus in cells expressing RII␣ could be caused by increased binding and endocytosis of the toxin. However, no significant stimulation of the binding or the endocytosis of the toxin after 30 min or 2 h of incubation were observed in the RII␣-expressing cells, strongly indicating a selective regulatory role of RII␣ in the transport of ricin from the endosomal compartments to the Golgi apparatus and further to the ER.
At the moment, we can only speculate about the molecular mechanism of the regulation of the endosome to Golgi transport of ricin by PKA type II␣. There are several examples of the importance of phosphorylation for transport. For example, it has been shown that the activity of PKA has an effect on the in vitro association of ARF1 to Golgi membranes (28). It is possible that upon stimulation, the Golgi-associated PKA type II␣  D to F, merged images of A and C, B and C, and A and B, respectively. Lymphoid Reh cells stably transfected with RII␣ were incubated with Cy5-labeled ricin (ϳ1000 ng/ml) for 30 min at 37°C. The cells were then fixed, permeabilized, stained with antibodies against RII␣ and a medial Golgi marker (CTR 433), and analyzed in a confocal microscope.
FIG. 9. Model of ricin endosome to Golgi transport. There is a well-established Rab9-dependent transport from late endosomes (LE) to the Golgi apparatus of furin and the M6PR. TGN38, Shiga toxin B, and ricin seem to be transported to the Golgi apparatus from early/ recycling endosomes (EE/RE). As indicated, several molecules implicated in the transport of ricin from the endosomal compartment to the Golgi apparatus have been identified. phosphorylates membrane proteins in neighboring compartments that recruit cytosolic proteins involved in the trafficking of ricin between endocytic organelles and the Golgi apparatus. Another possibility is that PKA type II␣ is important for the fusion of incoming vesicles with the Golgi apparatus.
Transport of ricin not only to the TGN, where the sulfotransferase is located (29), but also from the Golgi apparatus to the ER, measured as ricin that has been both sulfated and glycosylated, was shown to increase in cells expressing RII␣ relatively to the control cells in the presence of 8CPT-cAMP. An increased amount of glycosylated ricin could have been a result of the stimulated transport to the Golgi apparatus of this toxin. However, the fraction of ricin that has been both sulfated and glycosylated compared with the total amount of sulfated ricin is also increased in cells expressing RII␣ in the presence of 8CPT-cAMP, thus indicating an additional regulatory role of RII␣ in the transport of ricin from the Golgi to the ER.
In conclusion, our results indicate that the Golgi-associated type II␣ regulatory subunit of PKA regulates endosome-to-Golgi and Golgi-to-ER transport of ricin in lymphocytes.