Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines.

We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

Differential trafficking of the Niemann-Pick C1 and 2 proteins highlights distinct roles in late endocytic lipid trafficking.

UNLABELLED: The cellular location of Niemann-Pick C2 protein (NPC2) in cultured human fibroblasts and Chinese hamster ovary cells was examined immunocytochemically and in living cells by expression of a functional red fluorescent protein chimeric analogue. RESULTS: NPC2 is present in the lysosomes of both cholesterol-depleted and -replenished cells, unlike Niemann-Pick C1 protein (NPC1) which is recruited to late endosomes only upon uptake of low-density lipoprotein. With mobilization of cholesterol from lysosomes, immunocytochemical detection of NPC2 in lysosomes is greatly diminished, whereas NPC1 remains in the late endosomal compartment. We found a partial overlap in the trafficking and organellar sites of accumulation of NPC2 and NPC1. In living cells, NPC2 traffics with NPC1 in late endosomal tubules. However, in contrast to NPC1, which remains either in late endosomal vesicles and tubules or at the peripheries of cholesterol-laden lysosomes, NPC2 moves into the central core of lysosomes. Glycolipid analysis reveals that, in contrast to null mutant NPC1 cells, which accumulate GM2 ganglioside only at the plasma membrane, with no endocytic storage, absence of NPC2 protein in null mutant NPC2 cells does not block internalization of GM2 into endocytic vesicles. This difference in the cellular distribution of GM2 in NPC1 and NPC2 null mutants is the first report of a variation in the phenotypic expression of these genotypically distinct lesions. CONCLUSION: We speculate that while NPC1 may play a major role in the sorting of glycolipids as well as cholesterol within the late endosomes, NPC2 primarily plays a role in the egress of cholesterol and, potentially, glycolipids from lysosomes. These proteins appear not to be integrated into a tightly bound biological complex, but rather represent separate functional entities that complement each other.

The Stat3/5 locus encodes novel endoplasmic reticulum and helicase-like proteins that are preferentially expressed in normal and neoplastic mammary tissue.

The signal transducers and activators of transcription (STAT) 5 and 3 are critical for mammary alveolar development during pregnancy and remodeling during involution. In the mouse, STAT3, STAT5a, and STAT5b are encoded by adjacent genes on chromosome 11 (60.5 cM). To identify additional genes in the Stat3/5 locus that may participate in normal and neoplastic development of the mammary gland, we have cloned and sequenced 500 kb and searched for genes preferentially expressed in mammary tissue. We identified six known genes and cloned two new genes, termed D11Lgp1 and D11Lgp2. Both genes are most highly expressed in normal mammary tissue and mammary tumors from several transgenic mouse models. LGP1 consists of 532 and 530 amino acids in mouse and human, respectively (88% similarity). A region in the carboxy-terminal half of LGP1 has limited homology with Arabidopsis thaliana GH3-like proteins. Immunofluorescence studies demonstrated that LGP1 is located in the nuclear envelope and the endoplasmic reticulum. LGP2 is a cytoplasmic protein of 678 amino acids.

An isoform of branched-chain aminotransferase is a novel co-repressor for thyroid hormone nuclear receptors.

The functions of thyroid hormone receptors (TRs) are regulated by a host of co-regulatory proteins. Tissue-specific expression of these co-regulators leads to distinct expression patterns and regulation of thyroid hormone (T3) target genes in tissues. Previously we have found that human colon carcinoma RKO cells exhibit strong T3-independent transcriptional activity. We therefore searched for co-regulatory proteins in RKO cells using a yeast two-hybrid system with the intact TRbeta1 as bait. One of the three positive clones, designated as P3, was identified to be an isoform of human mitochondria branched-chain aminotransferase (BCATm). P3 was a spliced variant of BCATm with an internal 12-amino acid deletion near the carboxyl-terminal region and was abundantly expressed in RKO cells. The expressed protein localized both to the mitochondria and the nucleus of transfected CV1 cells. P3 physically interacted with TRbeta1 in a T3-independent manner that led to the inhibition in binding of TRbeta1 to thyroid hormone-responsive element. P3 not only enhanced the repressor activity of the unliganded TR but also repressed the ligand-dependent activation of TR. This repression was reversed by treatment of cells with trichostatin A, suggesting that in addition to the inhibition of DNA binding, the repression activity of P3 on TR may also be mediated by histone deacetylase activity. Thus, unlike the currently known co-repressors, P3 is a novel ligand-independent co-repressor for TR.

Glycan-dependent signaling: O-linked N-acetylglucosamine.

The addition of O-linked N-acetylglucosamine (O-GlcNAc) to target proteins may serve as a signaling modification analogous to protein phosphorylation. Like phosphorylation, O-GlcNAc is a dynamic modification occurring in the nucleus and cytoplasm. Various analytical methods have been developed to detect O-GlcNAc and distinguish it from glycosylation in the endomembrane system. Many target molecules have been identified; these targets are typically components of supramolecular complexes such as transcription factors, nuclear pore proteins, or cytoskeletal components. The enzymes responsible for O-GlcNAc addition and removal are highly conserved molecules having molecular features consistent with a signaling role. The O-GlcNAc transferase and O-GlcNAcase are likely to act in consort with kinases and phosphatases generating various isoforms of physiological substrates. These isoforms may differ in such properties as protein-protein interactions, protein stability, and enzymatic activity. Since O-GlcNAc plays a critical role in the regulation of signaling pathways of higher plants, the glycan modification is likely to perform similar signaling functions in mammalian cells. Glucose and amino acid metabolism generates hexosamine precursors that may be key regulators of a nutrient sensing pathway involving O-GlcNAc signaling. Altered O-linked GlcNAc metabolism may also occur in human diseases including neurodegenerative disorders, diabetes mellitus and cancer.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Calreticulin Is a receptor for nuclear export.

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739-14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes.

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.

Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export.

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

Hormone binding induces rapid proteasome-mediated degradation of thyroid hormone receptors.

The thyroid hormone 3,3',5-triiodo-l-thyronine (T3) is essential for growth, differentiation, and development. Its biological activities are mediated by T3 nuclear receptors (TRs). At present, how T3 regulates TR proteins and the resulting functional consequences are still unknown. Immunofluorescence analyses of endogenous TR in the growth hormone-producing GC cells showed that the T3-induced rapid degradation of TR was specifically blocked by lactacystin, a selective inhibitor of the ubiquitin-proteasome degradation pathway. Immunoblots demonstrated that the transfected TRbeta1 was ubiquitinated and that the ubiquitination was T3 independent. Studies with a series of truncated TRbeta1 showed that the hormone-binding domain was sufficient for the T3-induced rapid degradation of TRbeta1 by the proteasome degradation pathway. T3 also induced rapid degradation of TRbeta2 and TRalpha1. In contrast, the stability of the non-T3-binding TRalpha2 and naturally occurring TRbeta1 mutants that do not bind T3 was not affected by T3 treatment, indicating that hormone binding to receptor was essential for the degradation of the wild-type receptors. In the presence of proteasome protease inhibitors, the levels of both total and ubiquitinated TRbeta1 protein increased, yet T3-dependent transcriptional activation and the expression of the growth hormone gene were diminished, suggesting that proteasome-mediated degradation played a novel role in modulating transcriptional activation by TR. The present study reveals a role of T3 in modulating the functions of TR by regulating its receptor level via the ubiquitin-proteasome degradation pathway.

The long signal peptide isoform and its alternative processing direct the intracellular trafficking of interleukin-15.

Two isoforms of interleukin (IL)-15 exist: one with a short and another with a long signal peptide (LSP). Experiments using combinations of the LSP and mature proteins IL-2, IL-15, and green fluorescent protein revealed complex pathways of intracellular trafficking. In one pathway, the LSP was unprocessed, and IL-15 was not glycosylated, remained in the cytoplasm, and was degraded. The second trafficking pathway involved endoplasmic reticulum entry, N-linked glycosylation, and alternative partial LSP processing. The third pathway involved endoplasmic reticulum entry, followed by glycosylation, complete processing, and ultimately secretion. The complex intracellular trafficking patterns of LSP-IL-15 with its impediments to secretion as well as impediments to translation may be required due to the potency of IL-15 as an inflammatory cytokine. In terms of a more positive role, we propose that intracellular infection may relieve the burdens on translation and intracellular trafficking to yield effective IL-15 expression.

Dendrimer-assisted patch-clamp sizing of nuclear pores.

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be approximately equal to 10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5-10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Starburst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions.

Calcium, ATP and nuclear pore channel gating.

Nuclear envelope (NE) cisternal Ca2+ and cytosolic ATP are required for nuclear-pore-complex-(NPC-) mediated transport of DNAs, RNAs, transcription factors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. The large ion conductance (gamma) activity of the NPC channel (NPCC) is blocked by the NPC monoclonal antibody mAb414, known to block MMT, and is also silenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. To test the role of Ca2+ and ATP in NPCC activity, we carried out the present patch-clamp study with the pipette attached to the outer NE membrane of nuclei isolated from cultured Dunning G prostate cancer cells. Our investigations demonstrate that in these isolated nuclei neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. However, when simultaneously applied to the bath and pipette, they transiently silence NPCC activity through stimulation of MMT by raising the Ca2+ concentration in the NE cisterna ([Ca2+]NE). Our fluorescence microscopy observations with nuclear-targeted macromolecular fluorochromes (B-phycoerythrin and plasmid for the enhanced green fluorescence protein EGFP, pEGFP-C1) and with FITC-labeled RNA support the view that channel silence accompanies MMT. Repeated Ca2+ loading of the NE with Ca2+ and ATP, after unloading with 1-5 microM inositol 1,4,5-trisphosphate (IP3), thapsigargin (TSG) or 5 mM BAPTA or EGTA, failed to affect channel gating. This result indicates that other factors are involved in this phenomenon and that they are exhausted during the first cycle of NE Ca2+ loading/unloading--in agreement with current theories of NPC-mediated MMT. The results explain how Ca2+ and IP3 waves may convert the NE into an effective Ca2+ barrier and, consequently, affect the regulation of gene activity and expression through their feedback on MMT and NPCC gating. Thus, [Ca2+]NE regulation by intracellular messengers is an effective mechanism for synchronizing gene activity and expression to the cellular rhythm.

Organization of the mouse ASGR1 gene encoding the major subunit of the hepatic asialoglycoprotein receptor.

The hepatic asialoglycoprotein receptor was the first of the mammalian lectins to be recognized and has been the subject of intense investigation for three decades. Yet, the precise biological role of this major hepatic endocytic receptor has remained elusive. We describe here the characterization of the mouse gene for the major subunit of this receptor (ASGR1) along with 3.5 kb of the upstream 5' region. The gene comprises eight coding exons, with the major transcript in liver displaying a single non-coding 5' exon. A minor hepatic transcript initiates 435 bp upstream of the major start and includes an additional 5' non-coding exon and intron. A minimal 600 bp proximal region of ASGR1 exhibits hepatic-specific promoter activity in HepG2 cells in vitro. These results provide the basis for more detailed genetic studies on the functional role of the hepatic asialoglycoprotein receptor in mammals.

Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity.

O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors. In Arabidopsis, the OGT homolog participates in the gibberellin signaling pathway. We and others have proposed that mammalian OGT is the terminal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance. To facilitate mutational analysis of OGT in the absence of competing endogenous activity, we expressed the 103-kDa human OGT in Escherichia coli. Kinetic parameters for the purified recombinant enzyme (K(m) = 1.2 microM for Nup 62; K(m) = 0.5 microM for UDP-GlcNAc) are nearly identical to purified mammalian OGT. Deletions in the highly conserved C terminus result in a complete loss of activity. The N-terminal tetratricopeptide repeat domain is required for optimal recognition of substrates. Removal of the first three tetratricopeptide repeats greatly reduces the O-GlcNAc addition to macromolecular substrates. However, this altered enzyme retains full activity against appropriate synthetic peptides. Autoglycosylation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis. Given its proposed role in modulating insulin action, OGT may modify kinases involved in this signaling cascade. Among the many kinases tested, OGT glycosylates glycogen synthase kinase-3 and casein kinase II, two enzymes critical in the regulation of glycogen synthesis.

Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae.

The supply of nitrogen regulates yeast genes affecting nitrogen catabolism, pseudohyphal growth, and meiotic sporulation. Ure2p of Saccharomyces cerevisiae is a negative regulator of nitrogen catabolism that inhibits Gln3p, a positive regulator of DAL5, and other genes of nitrogen assimilation. Dal5p, the allantoate permease, allows ureidosuccinate uptake (Usa(+)) when cells grow on a poor nitrogen source such as proline. We find that overproduction of Mks1p allows uptake of ureidosuccinate on ammonia and lack of Mks1p prevents uptake of ureidosuccinate or Dal5p expression on proline. Overexpression of Mks1p does not affect cellular levels of Ure2p. An mks1 ure2 double mutant can take up ureidosuccinate on either ammonia or proline. Moreover, overexpression of Ure2p suppresses the ability of Mks1p overexpression to allow ureidosuccinate uptake on ammonia. These results suggest that Mks1p is involved in nitrogen control upstream of Ure2p as follows: NH(3) dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --> DAL5. Either overproduction of Mks1p or deletion of MKS1 interferes with pseudohyphal growth.

Phosphorylation and glycosylation of nucleoporins.

The nuclear pore complex mediates macromolecular transport between the nucleus and cytoplasm. Many nuclear pore components (nucleoporins) are modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). Among its many functions, protein phosphorylation plays essential roles in cell cycle progression. The role of O-GlcNAc addition is unknown. Here, levels of nucleoporin phosphorylation and glycosylation during cell cycle progression are examined. Whereas nuclear pore glycoproteins are phosphorylated in a cell-cycle-dependent manner, levels of O-GlcNAc remain constant. The major nucleoporin p62 can be phosphorylated in vitro by protein kinase A and glycogen synthase kinase (GSK)-3alpha but not by cyclin B/cdc2 or GSK-3beta. The consensus sites of these kinases resemble sites which can be glycosylated by O-GlcNAc transferase. These data are consistent with a model that O-GlcNAc limits nucleoporin hyperphosphorylation during M-phase and hastens the resumption of regulated nuclear transport at the completion of cell division.

Elevated O-linked N-acetylglucosamine metabolism in pancreatic beta-cells.

High intracellular glucose concentrations increase flux though the hexosamine biosynthetic pathway, resulting in elevated UDP-N-acetylglucosamine (GlcNAc) concentrations. The nucleocytoplasmic enzyme O-linked N-acetylglucosaminyltransferase (OGT) uses UDP-GlcNAc as a donor to modify numerous critical substrates, including nuclear pore proteins and transcription factors. Here, we document (a) the overwhelming enrichment of pancreatic OGT transcripts in the beta-cells of the islets of Langerhans, (b) the physiologically significant increase in the level of O-GlcNAc residues present in beta-cells, and (c) the action of streptozotocin, a close analogue of GlcNAc, to selectively inhibit O-GlcNAcase, an enzyme involved in the removal of O-GlcNAc residues. Taken together, these findings suggest that pancreatic beta cells maintain a highly elevated O-GlcNAc metabolism and that the diabetes inducing drug streptozotocin inhibits O-GlcNAcase.