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S Ponnambalam

Publications and source records attributed to S Ponnambalam.

At least 19 recordsLinked to original sources
PubMed1999

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

Animals↗
PubMed1998

A Golgi localization signal identified in the Menkes recombinant protein.

Menkes disease arises from a genetic impairment in copper transport. The gene responsible for the phenotype has been identified as a copper transporting ATPase ( ATP7A ). Recently, the protein encoded by the ATP7A gene has been localized to the Golgi complex. In order to investigate the role of the Menkes disease protein in copper transport, recombinant constructs containing both the full-length open reading frame and an alternatively spliced form have been successfully expressed and localized in mammalian cells. Other studies of a patient with occipital horn syndrome, an allelic variant of Menkes disease, have demonstrated that only this alternatively spliced isoform and not the full-length form is expressed in this patient. The milder form of this patient's phenotype suggests that the alternatively spliced isoform has some functional role in copper transport. In the present study the full-length recombinant Menkes protein was shown by immunofluorescence to localize to the Golgi apparatus and the alternatively spliced form, lacking sequences for transmembrane domains 3 and 4 encoded by exon 10, was shown to localize to the endoplasmic reticulum. Using sequences from exon 10 fused to a non-Golgi reporter molecule, a 38 amino acid sequence containing transmembrane domain 3 of the Menkes protein was found to be sufficient for localization to the Golgi complex. Therefore, the protein sequence encoded by exon 10 may be responsible for this differential localization and both isoforms may be required for comprehensive transport of copper within the cell.

Adenosine Triphosphatases↗
PubMed1997

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein.

The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2] [3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.

Androstadienes↗
PubMed1997

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.

Amino Acid Sequence↗
PubMed1997

Distinct compartmentalization of TGN46 and beta 1,4-galactosyltransferase in HeLa cells.

The Golgi proteins, TGN46 and GalT, were characterized in human HeLa cells using specific polyclonal and monoclonal antibodies. A bacterially expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN46 had an apparent molecular mass of 100 to 120 kDa which reflects extensive glycosylation. Epifluorescence light microscopy indicated substantial colocalization of TGN46 and GalT. However, confocal laser microscopy and three-dimensional reconstruction of double-labeled HeLa cells revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approximately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was detected in tubules. Distinct GalT-positive regions within the Golgi cisternal stack were not labeled for TGN46.

Animals↗
PubMed1996

Sorting sweet sorting. Protein secretion.

Membrane-spanning, lectin-like proteins in the eukaryotic secretory pathway seem to operate quality-control checkpoints by fine tuning protein exit or retention within each subcompartment.

Animals↗
PubMed1996

Targeting signal and subcellular compartments involved in the intracellular trafficking of HLA-DMB.

Evidence suggests that peptide loading onto MHC class II molecules occurs in a specialized late endocytic compartment (MIIC) where HLA-DM predominantly resides and in which MHC class II transiently accumulates before transport to the cell surface. We examined the targeting signals and compartments involved in the intracellular trafficking of human HLA-DM by expressing hybrid molecules comprising the cytoplasmic domain of DMB and luminal and transmembrane domains of CD8 in HeLa cells. A tyrosine-based tetrapeptide motif present in the cytoplasmic domain of DMB targeted hybrid molecules to intracellular vesicles. Mutation of the tyrosine residue to alanine resulted in redistribution of hybrid molecules to the cell surface. Correct intracellular targeting of HLA-DM was crucial for normal function in B cells. Immunoelectron microscopy on ultrathin cryosections showed that CD8-DMB molecules accumulated in late endocytic compartments sharing characteristics with lysosomes, like MHC class II compartments in APCs. Thus far, the exit of DMB from the Golgi complex has not been elucidated. Interestingly, we found that although the mannose 6-phosphate receptor and CD8-DMB contain similar tyrosine signals, no co-localization was observed in the trans-Golgi network, suggesting that these proteins are differentially sorted at this site. Co-transfection of CD8-DMB, HLA-DR alpha, HLA-DR beta, and an invariant chain revealed that HLA-DR molecules accumulated together with CD8-DMB in these lysosomal compartments. The similarity of these lysosomal-like compartments in wild-type and transfected cells suggests that they are part of the normal endocytic pathway in non-APCs.

Amino Acid Sequence↗
PubMed1996

Primate homologues of rat TGN38: primary structure, expression and functional implications.

cDNAs encoding the human and macaque homologues of rat TGN38 have been cloned and sequenced. The proteins have a highly conserved N terminus (comprising the signal peptide) and C terminus (comprising part of the lumenal domain, the membrane spanning region and cytoplasmic tail) but vary in the other part of the lumenal domain, which contains the repeat region. Whereas rat TGN38 contains 6 tandem repeats of an 8mer, both primate proteins possess 14 tandem repeats of a 14mer sequence. The human protein, like rat TGN38, is localised primarily to the TGN but is present on the cell surface and returns via endosomes. This behaviour is consistent with conservation of the membrane spanning region and the cytoplasmic tail, which contain the retention and retrieval signals, respectively, for localisation in the TGN. The unexpected differences in the lumenal domain can best be rationalised by the fact that both types of repeat domains have most of the properties of mucins. We suggest that TGN38 homologues are mucin-like molecules that regulate membrane traffic to and from the TGN.

Amino Acid Sequence↗
PubMed1994

Chromosomal location and some structural features of human clathrin light-chain genes (CLTA and CLTB).

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to be encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.

Animals↗
PubMed1994

The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network.

The membrane-spanning and cytoplasmic domains of CD4 and CD8 were replaced by those of TGN38. After transient expression in HeLa cells, the location of the hybrid proteins was determined using immunofluorescence and quantitative immuno-electron microscopy, FACS analysis and metabolic labeling. The membrane-spanning domain was found to contain a signal that localized hybrid proteins to the TGN. This was in addition to the signal previously identified in the cytoplasmic domain (Bos, K., C. Wraight, and K. Stanley. 1993. EMBO (Eur. Mol. Biol. Organ) J. 12:2219-2228. Humphrey, J. S., P. J. Peters, L. C. Yuan, and J. S. Bonifacino. 1993. J. Cell Biol. 120:1123-1135. Wong, S. H., and W. Hong. 1993. J. Biol. Chem. 268:22853-22862). The different properties of these two signals suggest that each operates by a different mechanism.

Amino Acid Sequence↗
PubMed1993

Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.

Amino Acid Sequence↗
PubMed1991

Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics.

Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.

Amino Acid Sequence↗
PubMed1990

Conservation and diversity in families of coated vesicle adaptins.

The complete sequence of the beta adaptin subunit of the plasma membrane adaptor complex from coated vesicles has been elucidated. Complementary cDNA clones from human fibroblasts, rat lymphocytes, and bovine lymphocytes have been isolated, sequenced, and compared with each other and with beta adaptin sequences from rat brain (Kirchhausen, T., Nathanson, K.L., Matsui, W., Vaisberg, A., Chow, E.P., Burne, C., Keen, J.H., and Davis, A.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2612-2616). Surprisingly, the 937-amino acid beta adaptin polypeptide is totally conserved between species. This remarkable homology contrasts with the absence of significant sequence similarity between the alpha (Robinson, M.S. (1989) J. Cell Biol. 108, 833-842) and beta adaptins of the plasma membrane adaptor complex. Diversity within each adaptin family is created by the expression of different genes and by tissue-specific differential splicing. The structures of the beta and alpha adaptins can both be divided into two globular domains interconnected by a variable and potentially flexible stalk domain.

Adaptor Protein Complex alpha Subunits↗
PubMed1988

Functional analysis of different sequence elements in the Escherichia coli galactose operon P2 promoter.

Starting with a DNA fragment containing the galactose operon P2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P2 activity. The results show that specific sequences upstream of -32 are not important. Removal of the sequence 5'-CACA-3' from -32 to -28 reduces P2 activity by 50%: longer deletions to -16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between -32 and -16 at gal P2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P2 in the absence of -35 region sequences is dependent on the sequence TG just upstream of the -10 hexamer, TATACT: a mutation at -14 changing the TG sequence to TT totally inactivates P2. However, P2 activity can be restored if the consensus -35 region sequence TTGACA is cloned 17 bp upstream of the -10 hexamer. Thus, for transcription initiation, the -10 hexamer, TATACT, must 'cooperate' with upstream sequences that may be located either around -35 or -14.

Base Sequence↗
PubMed1987

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein.

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.

DNA-Directed RNA Polymerases↗
PubMed1987

Binding of Escherichia coli RNA polymerase to a promoter carrying mutations that stop transcription initiation.

The gal P2 promoter can be inactivated by point mutations located in the -10 hexamer sequence or immediately upstream from it. Mutations at either site reduce expression in vivo and prevent the formation, in vitro, of tight complexes with RNA polymerase that give a strong footprint and can initiate transcription. However, with a mutation upstream from the -10 region, RNA polymerase could still make a specific contact with gal promoter DNA as judged by interference with cleavage by restriction enzyme SfaNI at a site within the promoter. In contrast, with a mutation in the -10 hexamer sequence, RNA polymerase could not make this contact and does not interfere with restriction by SfaNI.

Base Sequence↗
PubMed1987

RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination.

Using purified E. coli RNA polymerase we have studied the transcription in vitro of a series of DNA fragments carrying two tandemly arranged promoters, where the corresponding transcription start points were separated by 263, 138, 83 and 78 base pairs. In the case where the transcription start points are 83 base pairs apart, there is an interaction between RNA polymerase molecules transcribing from the two promoters. This interaction results in premature termination of the upstream transcript at a precise site. We propose that this is the result of RNA polymerase transcribing from the upstream promoter bumping into polymerase at the downstream promoter. The interaction between the two polymerase molecules is crucially dependent on the distance between the two promoters.

DNA-Directed RNA Polymerases↗