PKC controls HGF-dependent c-Met traffic, signalling and cell migration

The growth factor/receptor pair HGF/c-Met exerts control on proliferation, morphogenesis and motility, and through overexpression and mutation is implicated in cancer. Here we have investigated the relationship between receptor signalling and traffic, and its control by specific PKC isotypes. It is shown that c-Met signalling to the ERK cascade occurs within endosomal compartments and that it is in this compartment that PKCepsilon specifically exerts its control on the pathway with the consequent accumulation of ERK in focal complexes. These events are clearly separated from the subsequent microtubule-dependent sorting of c-Met to its perinuclear destination, which is shown to be under the control of PKCalpha. Thus while it is shown that traffic to endosomes is essential for HGF/c-Met to trigger an ERK response, the subsequent traffic and signalling of c-Met controlled by these two PKC isotypes are unconnected events. The dynamic properties conferred by the PKCepsilon control are shown to be essential for a normal HGF-dependent migratory response. Thus PKCs are shown to control both receptor traffic and signal traffic to relay HGF/c-Met responses.     

Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway

Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2β1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2β1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2β1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.     

PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.     

Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C₆-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.     

Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER

Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.     

Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex

We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.     

The endocytic pathway followed by the keratinocyte growth factor receptor

Keratinocyte growth factor (KGF/FGF7) acts specifically on epithelial cells and regulates their proliferation and differentiation. It binds to and activates a receptor tyrosine kinase, the KGF receptor (KGFR), which is a splicing variant of the fibroblast growth factor receptor 2. The endocytic pathway followed by KGF and its receptor was analyzed here using immunofluorescence and confocal microscopy. After 10 min of internalization at 37 degrees C, both KGF and its receptor were localized in early endosomes, and after 30-60 min of endocytosis ligand and receptor were seen to reach perinuclear late endosomes and not the recycling endosomal compartment. Parallel western blot analysis revealed that KGFRs were tyrosine phosphorylated both at early and late steps of internalization, suggesting that KGF and KGFR remain associated in active complexes through the endocytic pathway. Pulse-chase experiments showed that the internalized KGFRs underwent degradation detectable at 1 h of endocytosis at 37 degrees C, indicating that KGFRs are functionally downregulated.     

The Scribble Polarity Protein Stabilizes E-Cadherin/p120-Catenin Binding and Blocks Retrieval of E-Cadherin to the Golgi

Several polarity proteins, including Scribble (Scrb) have been implicated in control of vesicle traffic, and in particular the endocytosis of E-cadherin, but through unknown mechanisms. We now show that depletion of Scrb enhances endocytosis of E-cadherin by weakening the E-cadherin-p120catenin interaction. Unexpectedly, however, the internalized E-cadherin is not degraded but accumulates in the Golgi apparatus. Silencing p120-catenin causes degradation of E-cadherin in lysosomes, but degradation is blocked by the co-depletion of Scrb, which diverts the internalized E-cadherin to the Golgi. Loss of Scrb also enhances E-cadherin binding to retromer components, and retromer is required for Golgi accumulation of Scrb, and E-cadherin stability. These data identify a novel and unanticipated function for Scrb in blocking retromer-mediated diversion of E-cadherin to the Golgi. They provide evidence that polarity proteins can modify the intracellular itinerary for endocytosed membrane proteins.     

The long and the short cycle. Alternative intracellular routes for trafficking of G-protein-coupled receptors

The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in beta-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the "long cycle." The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane.     

Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment.

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC) mu involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC mu-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH(2)-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC mu completely abrogated Golgi localization of PKC mu. As an NH(2)-terminal PKC mu fragment was colocalized with p24, this region of PKC mu is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC mu to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKC mu found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC mu, in which Golgi compartment recruitment precedes and is essential for activation loop phosphorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH(2)-terminal serine(s) in the regulatory domain. PKC mu activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC mu function at the Golgi compartment.     

The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.     

Phylogenetic evidence for the distinction of Saaremaa and Dobrava hantaviruses

Background

Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi.

Results

We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide.

Conclusion

The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.     

The molecular mechanism of hepcidin-mediated ferroportin down-regulation

Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex Required for Transport proteins), by small-interfering RNA, reduces the trafficking of Fpn-green fluorescent to the lysosome.     

Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein

We have investigated the intracellular traffic of PrP(c), a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrP(c) is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrP(c), leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrP(c) trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrP(c) accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrP(c) but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrP(c) internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrP(c) is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrP(c) is delivered to classic endosomes after internalization.     

Functional role of the charge at the T538 residue in the control of protein kinase Cθ

We show that protein kinase C (PKC) θ localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCθ, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCθ does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCθ molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCθ.     

Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes

Entry of duck hepatitis B virus (DHBV) is initiated by specific interaction of its large envelope protein (L) with a cellular entry receptor, recently identified as carboxypeptidase D (CPD; historically gp180). In this report, we present evidence demonstrating that this receptor is down-regulated as a result of DHBV infection: (i) receptor levels determined by Western blot were much reduced in DHBV-infected duck livers and undetectable by immunostaining in infected cultured hepatocytes; (ii) results from metabolic labeling experiments indicate enhanced receptor protein turnover; (iii) the kinetics of receptor loss from newly infected cells correlated with the accumulation of newly synthesized viral protein; (iv) expression of DHBV L protein, transduced from a recombinant adenovirus, was sufficient to eliminate gp180/CPD from the Golgi compartment, its normal predominant location; (v) gp180/CPD remained absent from the Golgi compartment in infected hepatocytes, even after overexpression from a recombinant adenovirus, while residual amounts subsequently became detectable in a perinuclear compartment, containing DHBV L protein; (vi) expression of DHBV L protein in a HepG2 cell line, stably expressing gp180/CPD, leads to incomplete receptor maturation and induces its degradation. Taken together, these data are consistent with a model in which the virus receptor interacts early in the biosynthetic pathway with the viral L protein, leading to its retention in a pre-Golgi compartment and to subsequent degradation, thus preventing receptor interference with the export of DHBV via the secretory pathway which it shares with its receptor. Accordingly, and analogously with receptor down-regulation in retroviral systems, DHBV receptor down-modulation may account for the much-reduced efficiency of DHBV superinfection of preinfected hepatocytes.     

Important role for the V-type H(+)-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.     

Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways.

Toxoplasma gondii actively penetrates its vertebrate host cell to establish a nonfusigenic compartment called the parasitophorous vacuole (PV) that has previously been characterized primarily in phagocytic cells. To determine the fate of this unique compartment in nonphagocytic cells, we examined the trafficking of host cell proteins and lipids in Toxoplasma-infected fibroblasts using quantitative immunofluorescence and immunoelectron microscopy. Toxoplasma-containing vacuoles remained segregated from all levels of the endocytic pathway, as shown by the absence of delivery of transferrin receptors, mannose phosphate receptors, and the lysosomal-associated protein LAMP1 to the vacuole. The PV was also inaccessible to lipids (DiIC16, and GM1) that were internalized from the plasma membrane via the endocytic system. In contrast, vacuoles containing dead parasites or zymosan sequentially acquired both endosomal and lysosomal protein markers and host lipids, reflecting the competency of fibroblasts to process phagocytic vacuoles. The mature PV often lies adjacent to the host cell Golgi, suggesting that it may intersect with vesicles from the exocytic pathway. Despite this proximity, the PV was inaccessible to nitrobenzadiazole-labeled sphingolipids exported from the Golgi and did not contain the host protein markers AP1 or beta-COP. Our results demonstrate that Toxoplasma resides in a compartment that excludes delivery of protein and lipid components from the host cell endocytic and exocytic pathways.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.