Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin-coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.     

The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum

In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.     

Characterization of KIF1C, a new kinesin-like protein involved in vesicle transport from the Golgi apparatus to the endoplasmic reticulum

Kinesins comprise a large family of microtubule-based motor proteins, of which individual members mediate specific types of motile processes. Using the ezrin domain of the protein-tyrosine phosphatase PTPD1 as a bait in a yeast two-hybrid screen, we identified a new kinesin-like protein, KIF1C. KIF1C represents a member of the Unc104 subfamily of kinesin-like proteins that are involved in the transport of mitochondria or synaptic vesicles in axons. Like its homologues, the 1103-amino acid protein KIF1C consists of an amino-terminal motor domain followed by a U104 domain and probably binds to target membranes through carboxyl-terminal sequences. Interestingly, KIF1C was tyrosine-phosphorylated after peroxovanadate stimulation when overexpressed in 293 or NIH3T3 fibroblasts or in native C2C12 cells. Using immunofluorescence, we found that KIF1C is localized primarily at the Golgi apparatus. In brefeldin A-treated cells, the Golgi membranes and KIF1C redistributed to the endoplasmic reticulum (ER). This brefeldin A-induced flow of Golgi membranes into the ER was inhibited in cells transiently overexpressing catalytically inactive KIF1C. In conclusion, our data suggest an involvement of tyrosine phosphorylation in the regulation of the Golgi to ER membrane flow and describe a new kinesin-like motor protein responsible for this transport.     

Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo

Experimental glomerulonephritis was induced in rats to investigate the consequence of the antigen-antibody interaction on the surface of glomerular endothelial cells (GENs). A lectin, Lens culinaris hemoagglutinin (LCH), was first planted in the left kidney by isolated perfusion of a left kidney, and then the circulation was reestablished. Rabbit anti-LCH antibodies were injected from the tail vein 3 minutes after the recirculation of the left kidney, and acute glomerulonephritis ensued. Fifteen minutes after the injection, rabbit immunoglobulin G (IgG), rat C3, and LCH were observed exclusively on the surface of GENs. Accumulation of platelets was prominent. Three hours later, the immune deposits were seen in the subendothelial space, and the polymorphonuclear cells were the dominant infiltrate in the glomeruli. Up to the seventh day, immune deposits were seen in the subendothelial space, and the widening of this area was increasingly observed. Fourteen days later, immune deposits containing rat IgG were observed in the subepithelial area, but they were only occasionally seen in the subendothelial space and in the mesangial area. No crescent formation was seen at day 14, but the mesangial area was expanded, with an increased number of cells. The number of nuclei in the cross-section of a glomerulus increased after the induction of glomerulonephritis, but the number of leukocyte common antigen-positive cells (infiltrating cells) decreased gradually from day 4 to day 14. The staining of Thy-1.1, a marker of mesangial cell, was markedly enlarged in the glomerulus at day 14. These data suggest that mesangial proliferative glomerulonephritis can be induced by the antigen-antibody interaction on the surface of GENs.     

The relationship of the endoplasmic reticulum to the Golgi complex in Tetrahymena pyriformis

The endoplasmic reticulum (ER) and the closely connected, single dictyosomal Golgi apparatus of Tetrahymena pyriformis cells showed random distribution in the cytoplasm. Ribosomes were evident, and coated vesicles pinched off from the ER were seen. The membranes of the endoplasmic reticulum generally formed a tube-like structure, although after histamine treatment multiple, folded and circular structures were observed. The number of coated vesicles detaching from the endoplasmic reticulum increased as a result of histamine treatment.     

Dynamic recycling of ERGIC53 between the endoplasmic reticulum and the Golgi complex is disrupted by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, has recently been demonstrated to block protein transport from the endoplasmic reticulum (ER) to the Golgi complex. The ER to Golgi transport is primarily operated by the ER-Golgi intermediate compartment (ERGIC). We examined the effect of NDGA on the ERGIC, focusing on the distribution of its marker ERGIC53. In control cells ERGIC53 was distributed to vesicular tubular structures corresponding to the ERGIC as well as to the ER and the cis-Golgi, reflecting its cycling between these compartments. Upon treatment of cells with NDGA, ERGIC53 was rapidly accumulated in the Golgi and undetectable in the ER and the ERGIC. Prolonged incubation of cells with the drug, however, caused redistribution of ERGIC53 and resident Golgi proteins to the ER. Thus, it is likely that NDGA has dual effects on ERGIC53 cycling; the initial accumulation in the Golgi may be caused by blocking its retrieval from the cis-Golgi to the ER/ERGIC, while the delayed redistribution to the ER may occur through a pathway induced by the drug that is different from the COPI-dependent pathway.     

MUC4 is a major component of salivary mucin MG1 secreted by the human submandibular gland

High molecular weight salivary mucin (MG1) is an important component of saliva, contributing to the lubricative and tissue-protective functions of this biological fluid. We have shown previously that the human mucin gene MUC5B is expressed at high levels in sublingual gland and is a significant constituent of MG1. Since many epithelia express multiple mucin genes, it seemed likely that MG1 in salivary secretions is also a heterogeneous mixture of mucin gene products. The aim of this study was to determine whether MUC4, a mucin shown in Northern blotting experiments to be expressed in salivary glands, was a significant protein component of MG1 in salivary secretions. Two cDNA clones containing MUC4 tandem repeats were isolated from a human submandibular gland cDNA library. In addition, recombinant MUC4 produced in a bacterial expression system cross-reacted with an antibody directed against deglycosylated MG1. This shows conclusively that human salivary mucin MG1 contains both MUC5B and MUC4 gene products suggesting that each mucin may perform distinct functions in the oral cavity.     

Significance of MUC1 and MUC2 mucin expression in colorectal cancer

AIMS: To compare the lineage specific distribution of MUC1 and MUC2 mucins in normal colorectal mucosa and adenocarcinoma and to identify pathological correlations. METHODS: Paraffin wax sections from 51 colorectal cancers were examined for the expression of MUC1 and MUC2, non-O-acetyl sialic acid and the carbohydrate epitopes Lex, Ley, sialosyl-Lex, sialosyl-Tn, and Tn using standard histochemical methods. RESULTS: MUC1, Lex and Ley co-localised with columnar cell secretions, whereas MUC2, mild periodic acid Schiff and sialosyl-Tn co-localised with goblet cell mucin in both normal and malignant tissues. Sialosyl-Lex and Tn were associated with both lineages. In normal tissues MUC1, Lex and Ley showed only trace expression by crypt base columnar cells. Cancers could be classified into four phenotypes (MUC2+/MUC1-, MUC2+/MUC1+, MUC2-/MUC1+, MUC2-/MUC1-). Particular phenotypes showed significant correlations with cancer type, lymph node spread and peritumoral lymphocytic infiltration and trends falling short of significance in relation to grade of differentiation and contiguous adenoma. CONCLUSIONS: Classification of colorectal cancer by means of lineage specific function may be relevant to both pathogenesis and prognosis.     

Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus

The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.     

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.     

Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca2+) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca2+ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca2+-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca2+ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca2+ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca2+ pool in untreated cells when extracellular Ca2+ is low. However, low extracellular Ca2+ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca2+ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca2+ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Oncogenic activation of a human cyclin A2 targeted to the endoplasmic reticulum upon hepatitis B virus genome insertion

Cyclins are major cell cycle regulators which role in malignant transformation remains controversial. In this report we describe a new mechanism of cyclin oncogenic activation. We demonstrate that an altered form of cyclin A2 (S2A) which N-terminal part is replaced by the hepatitis B virus envelope protein transforms normal rat kidney cells and cooperates with ras to transform rat embryo fibroblasts. In contrast, neither the viral moiety, nor a full length or N-terminally deleted cyclin A2 show these oncogenic properties. S2A oncogenicity arises from its binding to cyclin dependent kinases, since mutation in the MRAIL sequence abolishes transformation and correlates with an abnormal cellular localization in the endoplasmic reticulum membrane. Together, these results implicate modification in the cellular distribution of a cell cycle regulator as a mechanism of virally-induced transformation.     

Substrate specificities of recombinant murine Golgi alpha1, 2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2-cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.     

Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport

COPII-coated endoplasmic reticulum (ER)-derived transport vesicles contain a distinct set of membrane-bound polypeptides. We have obtained the NH2-terminal amino acid sequence of polypeptide constituents found on purified vesicles and in this report investigate the 24- and 25-kDa species. The 24-kDa protein is identical to Emp24p, a type I transmembrane protein that is required for transport of a subset of secretory proteins from the ER to the Golgi complex (Schimm?ller, F., Singer-Krüger, B., Schr?der, S., Krüger, U., Barlowe, C., and Riezman, H. (1995) EMBO J. 14, 1329-1339). The 25-kDa protein, termed Erv25p (ER vesicle protein of 25 kDa), corresponds to an open reading frame found on chromosome XIII of Saccharomyces cerevisiae. Erv25p shares overall sequence identity with Emp24p, but the two proteins are not functionally interchangeable. Antibodies directed against Erv25p reveal that Emp24p and Erv25p depend on each other for stability and form a protein complex that can be isolated after chemical cross-linking. Yeast strains lacking Erv25p (erv25Delta) are viable and display the same selective defect in transport of secretory proteins from the ER to Golgi complex as an emp24Delta strain. A cell-free assay that measures vesicle formation from ER membranes demonstrates that Erv25p and Emp24p are incorporated equally into ER-derived vesicles when COPII-coated budding is reconstituted. Vesicle formation from an erv25Delta strain, an emp24Delta strain and a double erv25Delta emp24Delta strain proceed at wild-type levels; however, incorporation of the Erv25p or the Emp24p protein into COPII-coated vesicles requires expression of both subunits. A potential model for transport of the Erv25p-Emp24p complex between the ER and Golgi compartments is discussed.     

Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus

The objective of this study was to determine the predictability of endosseous implants placed in a maxillary sinus grafted with a mixture of bovine porous bone mineral and demineralized freeze-dried bone. Sixty implants were placed in 20 patients representing 28 sinuses using either a one- or two-stage technique. After an implant loading period of more than 2 years, the survival rate (eg, a clinically functioning implant without signs of mobility or infection) varied from 90% to 96%. No infections or other complications were encountered. The data suggest that this treatment regimen can result in a high rate of survival.     

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.     

Characterization of AtSEC12 and AtSAR1. Proteins likely involved in endoplasmic reticulum and Golgi transport

Transport of cargo proteins from the endoplasmic reticulum (ER) to the cis-Golgi network is mediated by protein-coated vesicles. The coat, called COPII coat, consists of proteins that are recruited from the cytosol and interact with integral membrane proteins of the ER. In yeast, both cytosolic proteins (Sec13/31, Sec23/24, and Sar1) and ER-associated proteins (Sec12 and others) have been purified and characterized and it has been possible to demonstrate transport vesicle formation in vitro. Arabidopsis thaliana homologs of Sar1 and Sec12 have recently been identified, but little is known about the properties of the proteins or their subcellular distribution. Here we demonstrate that AtSAR1, a 22-kD protein that binds GTP, and AtSEC12, a 43-kD GTP-exchange protein, are both associated with the ER. However, about one-half of the cellular AtSAR1 is present in the cytosol. When AtSAR1 is overexpressed in transgenic plants, the additional protein is also cytosolic. When tissue-culture cells are cold-shocked (12 h at 8 degrees C), AtSAR1 levels appeared to decline and a larger proportion of the total protein was found in the cytosol. Given the known function of AtSAR1 in yeast, we propose that the amount of ER-associated AtSAR1 is an indication of the intensity of the secretory process. Thus, we expect that such a cold shock will adversely affect ER-to-Golgi transport of proteins.     

Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal alpha1,2 glucose and the first alpha1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for misfolded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.     

Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells

The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane. In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines. Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region. Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus. The same observations have been obtained on a primary culture from a metastasis of human melanoma. Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined. MRP1 also showed Golgi-like localization. The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic. P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells. On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.