Altered regulation of P-450IA1 expression in a multidrug-resistant MCF-7 human breast cancer cell line

MCF-7 human breast cancer cells, selected for resistance to adriamycin (AdrR), exhibit the phenotype of multidrug resistance (MDR). Previous studies have shown that resistance in AdrR MCF-7 cells is associated with several biochemical changes that are similar to those induced in rat hyperplastic nodules, preneoplastic liver lesions which display broad spectrum resistance to carcinogens and hepatotoxins. In this report, we show that these changes in the AdrR MCF-7 cells are also associated with the development of cross-resistance to the procarcinogen benzo(a)pyrene (BP) and are associated with a marked defect in the conversion of BP to its cytotoxic, carcinogenic metabolites by AdrR cells. Since aryl hydrocarbon hydroxylase is the principle enzyme activity which converts benzo(a)pyrene to toxic hydroxylated forms, the regulation of cytochrome P-450IA1 expression, the gene encoding this enzyme activity in MCF-7 cells, was examined. Incubation with 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a marked increase in aryl hydrocarbon hydroxylase activity in wild type (WT) but not AdrR MCF-7 cells. The alteration in aryl hydrocarbon hydroxylase expression in the AdrR cells is not overcome by incubation either with higher concentrations of TCDD (1 microM) or for longer periods of time (4 days). Northern blot analysis indicates that this defect in AdrR MCF-7 cells involves a regulatory defect at the level of P-450IA1 RNA. Following transfection of a construct containing the normal mouse P-450IA1 promoter fused to a reporter gene (bacterial chloramphenicol acetyltransferase) into WT and AdrR MCF-7 cells, TCDD induced chloramphenicol acetyltransferase activity in WT MCF-7 cells only. Furthermore, TCDD also induces both DT-diaphorase and UDP-glucuronyltransferase activities in WT, but not AdrR cells. These data suggest that the defect in the AdrR MCF-7 cells is not due to a structural P-450IA1 gene mutation, but rather involves a product regulating the polycyclic hydrocarbon-inducible expression of several drug-metabolizing enzyme activities. This defect in the AdrR MCF-7 cells is also associated with the development of resistance to ellipticine, an anticancer agent which is converted to more toxic hydroxylated species by aryl hydrocarbon hydroxylase or a similar mixed function oxidase. The WT and AdrR MCF-7 cells represent a useful model to study the regulation of the P-450IA1 gene in human cells.     

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.     

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.     

The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin-based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi-associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin-based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS-7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin(195), but were accompanied by the disappearance of the well-focused microtubule-organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP-AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.     

GPHR is a novel anion channel critical for acidification and functions of the Golgi apparatus

The organelles within secretory and endocytotic pathways in mammalian cells have acidified lumens, and regulation of their acidic pH is critical for the trafficking, processing and glycosylation of cargo proteins and lipids, as well as the morphological integrity of the organelles. How organelle lumen acidification is regulated, and how luminal pH elevation disturbs these fundamental cellular processes, is largely unknown. Here, we describe a novel molecule involved in Golgi acidification. First, mutant cells defective in Golgi acidification were established that exhibited delayed protein transport, impaired glycosylation and Golgi disorganization. Using expression cloning, a novel Golgi-resident multi-transmembrane protein, named Golgi pH regulator (GPHR), was identified as being responsible for the mutant cells. After reconstitution in planar lipid bilayers, GPHR exhibited a voltage-dependent anion-channel activity that may function in counterion conductance. Thus, GPHR modulates Golgi functions through regulation of acidification.     

The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR.

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.     

Elevated Golgi pH impairs terminal N‐glycosylation by inducing mislocalization of Golgi glycosyltransferases

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N‐glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 µM CQ) is sufficient to markedly impair terminal α(2,3)‐sialylation of an N‐glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding α(2,3)‐sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair α(2,6)‐sialylation, or the proximal glycosylation steps such as β(1,4)‐galactosylation or acquisition of Endo H resistance, and the steady‐state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N‐glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases. J. Cell. Physiol. 220: 144–154, 2009. © 2009 Wiley‐Liss, Inc.     

Defective acidification of intracellular organelles results in aberrant secretion of cathepsin D in cancer cells

Aberrant secretion of lysosomal hydrolases such as (pro)cathepsin D (proCD) is a common phenotypic change in many human cancers. Here we explore the underlying molecular defect(s) and find that MCF-7 breast and CaCo-2 colorectal cancer cells that are unable to acidify their endosomal compartments secreted higher amounts of proCD than did acidification-competent cancer cell types. The latter secreted equivalent amounts of proCD only after dissipation of their organellar pH gradients with NH(4)Cl. Assessing the critical steps that resulted in proCD secretion revealed that the Golgi-associated sorting receptor for CD, i.e. the cation-independent mannose-6-phosphate receptor (MPR300), was aberrantly distributed in acidification-defective MCF-7 cells. It accumulated mainly in late endosomes and/or lysosomes as a complex with its ligand (proCD or intermediate CD), as evidenced by its co-localization with both CD and LAMP-2, a late endosome/lysosome marker. Our immunoprecipitation analyses also showed that MCF-7 cells possessed 7-fold higher levels of receptor-enzyme complexes than did acidification-competent cells. NH(4)Cl induced similar receptor redistribution into LAMP-2-positive structures in acidification-competent cells but not in MCF-7 cells. The receptor also recovered its normal Golgi localization upon drug removal. Based on these observations, we conclude that defective acidification results in the aberrant secretion of proCD in certain cancer cells and interferes mainly with the normal disassembly of the receptor-enzyme complexes and efficient receptor reutilization in the Golgi.     

Cog2 null mutant CHO cells show defective sphingomyelin synthesis

The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ～25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C(6)-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.     

Promotion of Cell Migration by Neural Cell Adhesion Molecule (NCAM) Is Enhanced by PSA in a Polysialyltransferase-Specific Manner

Neural cell adhesion molecule 140 (NCAM-140) is a glycoprotein and always highly polysialylated in cancer. Functions of polysialic acid (PSA) that binds to N-glycan termini on NCAM remain unclear. ldlD-14 cells, a CHO cell mutant deficient in UDP-Gal 4-epimerase, are useful for structural and functional studies of Gal-containing glycoproteins because their abnormal glycosylation can be converted to normal status by exogenous addition of galactose (Gal). We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7. The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied. We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. In addition, PSA catalyzed by different polysialyltransferases affected the adhesion of NCAM to different extracellular matrix (ECM) components.     

Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.     

Recombinant MUC1 probe authentically reflects cell-specific O-glycosylation profiles of endogenous breast cancer mucin. High density and prevalent core 2-based glycosylation

Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of the O-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75-1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with alpha 3-linked sialic acid (MDA-MB231, ZR75-1) or alpha 2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identical O-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming beta 6-N-acetylglucosaminyltransferases and/or to overexpression of competitive alpha 3- sialyltransferase.     

Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells

Adriamycin-resistant (AdrR) human breast cancer cells have been selected which exhibit cross-resistance to a wide range of anti-cancer drugs. This multidrug-resistant phenotype is associated with increases in the activities of glutathione peroxidase and glutathione transferase. The 45-fold increase in glutathione transferase activity is associated with the appearance of a new anionic isozyme in AdrR cells which is immunologically related to the anionic glutathione transferase present in human placenta. The increase in transferase and the level of drug resistance is relatively stable during passage of AdrR cells in the absence of adriamycin for over 10 months. A similar anionic glutathione transferase isozyme is also found in rat hyperplastic liver nodules, a preneoplastic state resulting from exposure to carcinogens. A rat cDNA which codes for the anionic glutathione transferase in rat hyperplastic nodules hybridizes to a 1.1-kilobase pair mRNA which is overexpressed in the AdrR MCF-7 cells. The anionic transferase has been purified from the AdrR cells and found to have characteristics which distinguish it from other anionic human glutathione transferases, including high levels of intrinsic peroxidase activity. The overexpression of a similar anionic glutathione transferase in human breast cancer cells selected for multidrug resistance and in rat hyperplastic liver nodules, which develop resistance to various hepatotoxins, suggests a possible role for this drug-conjugating enzyme in the mechanism of resistance in both of these states.     

C. elegans as a model system to study the function of the COG complex in animal development

The conserved oligomeric Golgi (COG) complex is an octameric protein complex associated with the Golgi apparatus and is required for proper sorting and glycosylation of Golgi resident enzymes and secreted proteins. Although COG complex function has been extensively studied at the cellular and subcellular levels, its role in animal development mostly remains unknown. Recently, mutations in the components of the COG complex were found to cause abnormal gonad morphogenesis in Caenorhabditis elegans. In C. elegans, the COG complex acts in the glycosylation of an ADAM (a disintegrin and metalloprotease) family protein, MIG-17, which directs migration of gonadal distal tip cells to lead gonad morphogenesis. This is the first link between the COG complex and the function of an ADAM protease that is directly involved in organ morphogenesis, demonstrating the potential of C. elegans as a model system to study COG function in animal development.     

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Background

Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).

Methodology/Principal Findings

Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.

Conclusions/Significance

Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.     

Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions

The glycoproteins of tumour cells are often abnormal, both in structure and in quantity. In particular, the mucin-type O-glycans have several cancer-associated structures, including the T and Tn antigens, and certain Lewis antigens. These structural changes can alter the function of the cell, and its antigenic and adhesive properties, as well as its potential to invade and metastasize. Cancer-associated mucin antigens can be exploited in diagnosis and prognosis, and in the development of cancer vaccines. The activities and Golgi localization of glycosyltransferases are the basis for the glycodynamics of cancer cells, and determine the ranges and amounts of specific O-glycans produced. This review focuses on the glycosyltransferases of colon and breast cancer cells that determine the pathways of mucin-type O-glycosylation, and the proposed functional and pathological consequences of altered O-glycans.     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase.