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1.
FEBS J ; 283(6): 1168-79, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26797772

ABSTRACT

Tissue-nonspecific alkaline phosphatase (TNSALP) is a membrane glycoprotein with a proposed role in bone mineralization. Indeed, mutations in TNSALP have been identified in patients with hypophosphatasia (HPP), a genetic disease characterized by hypomineralization of bone and teeth and a deficiency in serum ALP activity. TNSALP has five potential N-glycosylation sites at N140, N230, N271, N303 and N430 by standard nomenclature. A mutation at one of these sites, N430, was recently detected in a patient with infantile HPP. Using site-directed mutagenesis, we demonstrated that TNSALP has five N-glycans in transfected COS-1 cells and that individual single N-glycan deletion mutants of TNSALP retain the dimeric structure required for ALP activity, excluding the possibility that any single N-glycan plays a vital role in the structure and function of TNSALP. However, we found that TNSALP (N430Q) and TNSALP (N430E) mutants, but not a TNSALP (N430D) mutant, failed to form dimers. The TNSALP (N430S) mutant linked to infantile HPP was glycosylation-defective and unable to dimerise, similar to TNSALP (N430Q) and TNSALP (N430E) mutants; therefore, TNSALP (N430S) was established as a severe allele without strong ALP activity. By contrast to individual single N-glycan deletion mutants, TNSALP devoid of all five N-glycans was present to a much lesser extent than wild-type TNSALP in transfected cells, possibly reflecting its instability. A comprehensive analysis of a series of multiple N-glycan depletion mutants in TNSALP revealed that three N-glycans on N230, N271 and N303 were the minimal requirement for the structure and function of TNSALP and a prerequisite for its stable expression in a cell.


Subject(s)
Alkaline Phosphatase/chemistry , Alkaline Phosphatase/genetics , Hypophosphatasia/enzymology , Hypophosphatasia/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Alkaline Phosphatase/metabolism , Amino Acid Substitution , Animals , Binding Sites/genetics , COS Cells , Chlorocebus aethiops , Gene Expression Regulation, Enzymologic , Glycosylation , Humans , Infant , Mutagenesis, Site-Directed , Mutant Proteins/metabolism , Mutation, Missense , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
2.
Arch Biochem Biophys ; 587: 31-7, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26475675

ABSTRACT

Tumor necrosis factor-α (TNFα)-induced reactions are effective to maintain homeostasis; however, excessive responses play progressive roles in the pathogenesis of various chronic inflammatory diseases. We demonstrate that TNFα triggered the release of its receptor TNFR1 as a content of extracellular vesicles (EVs) from the human bronchial epithelial cell, BEAS-2b. The TNFR1 cytoplasmic domain binding partner, TNFR-associated death domain (TRADD), was released by TNFα treatment along with TNFR1. TNFα-triggered release of EVs was decreased in the presence of amitriptyline, an inhibitor of acid sphingomyelinase (A-SMase), or of GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), indicating that EVs containing TNFR1 and TRADD are released through A-SMase and N-SMase dependent manners. From sucrose density gradient analysis, each sphingomyelinase is involved in the generation of distinct populations of EVs. Inhibition of A-SMase or N-SMase resulted in significantly increased responses to TNFα in parental cells. Given that TRADD serves as a platform for the assembly of subsequent signaling molecules, the TNFα triggered release of TNFR1 and TRADD might be an effective strategy for down regulation of the TNFα responses of parental cells.


Subject(s)
Extracellular Vesicles/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Respiratory Mucosa/cytology , TNF Receptor-Associated Death Domain Protein/metabolism , Tumor Necrosis Factor-alpha/metabolism , Bronchi/cytology , Bronchi/metabolism , Cell Line , Humans , Respiratory Mucosa/metabolism , Sphingomyelin Phosphodiesterase/metabolism
3.
Mol Genet Metab ; 115(4): 180-5, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25982064

ABSTRACT

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-ß-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface.


Subject(s)
Alkaline Phosphatase/genetics , Hypophosphatasia/genetics , Leucine/metabolism , Mutation , Proline/metabolism , Tooth Demineralization/congenital , Alkaline Phosphatase/chemistry , Alkaline Phosphatase/metabolism , Animals , CHO Cells , COS Cells , Chlorocebus aethiops , Cricetulus , Humans , Hypophosphatasia/enzymology , Phenotype , Tooth Demineralization/enzymology , Tooth Demineralization/genetics
4.
FEBS J ; 282(11): 2232-44, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25787021

ABSTRACT

GM130 is a cytoplasmic peripheral membrane protein localized on the cis side of the Golgi apparatus. GM130 is proposed to function as a membrane skeleton, maintaining the structure of the Golgi apparatus, and as a vesicle tether that facilitates vesicle fusion to the Golgi membrane. More than 60% of the GM130 molecule is believed to exist as coiled-coil structures with a probability above 90%, based on its primary amino acid sequence. The predicted coiled-coil region was similar to that of yeast Uso1p and its mammalian homolog, p115, both of which form coiled-coil homodimers. Therefore, GM130 has long been thought to form a homodimer with a rod-like shape. However, our biochemical and electron microscopical analyses revealed that GM130 is a parallel homotetramer with a flexible rod-like structure with I- and Y-shaped conformations. The structure of the N-terminal region may interchange between an open conformation (branched or Y-shaped) and a closed conformation (non-branched or I-shaped), possibly with the help of interacting molecules. This conformational change may alter the oligomeric state of the GM130 molecules and the function of GM130 in the vesicle tethering and the maintenance of the Golgi structure.


Subject(s)
Autoantigens/chemistry , Membrane Proteins/chemistry , Animals , Autoantigens/ultrastructure , Humans , Membrane Proteins/ultrastructure , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Tertiary , Rats
5.
Biochem Biophys Res Commun ; 456(1): 275-81, 2015 Jan 02.
Article in English | MEDLINE | ID: mdl-25436429

ABSTRACT

p230/golgin-245 is a trans-Golgi coiled-coil protein that is known to participate in regulatory transport from the trans-Golgi network (TGN) to the cell surface. We investigated the role of p230 and its interacting protein, microtubule actin crosslinking protein 1 (MACF1), in amino acid starvation-induced membrane transport. p230 or MACF1 knock-down (KD) cells failed to increase the autophagic flow rate and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive puncta under starvation conditions. Loss of p230 or MACF1 impaired mAtg9 recruitment to peripheral phagophores from the TGN, which was observed in the early step of autophagosome formation. Overexpression of the p230-binding domain of MACF1 resulted in the inhibition of mAtg9 trafficking in starvation conditions as in p230-KD or MACF1-KD cells. These results indicate that p230 and MACF1 cooperatively play an important role in the formation of phagophore through starvation-induced transport of mAtg9-containing membranes from the TGN. In addition, p230 itself was detected in autophagosomes/autolysosome with p62 or LC3 during autophagosome biogenesis. Thus, p230 is an important molecule in phagophore formation, although it remains unclear whether p230 has any role in late steps of autophagy.


Subject(s)
Autoantigens/metabolism , Autophagy , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Actins/metabolism , Biological Transport , Cell Membrane/metabolism , Golgi Apparatus/metabolism , HeLa Cells , Humans , Microtubules/metabolism , Phagosomes/metabolism , Plasmids/metabolism , Protein Transport , RNA, Small Interfering/metabolism
6.
Epilepsy Res ; 108(3): 420-32, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24480790

ABSTRACT

Mutations in GABRG2, which encodes the γ2 subunit of GABAA receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABAA receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and ß2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABAA receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABAA receptor subunits α1 and ß2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and ß2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2.


Subject(s)
Codon, Nonsense/genetics , Epilepsy/genetics , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Animals , Brain/metabolism , Brain/pathology , Cells, Cultured , Child, Preschool , Epilepsies, Myoclonic/genetics , Epilepsies, Myoclonic/pathology , Female , Gene Expression Regulation , HEK293 Cells , Hippocampus/cytology , Humans , Infant , Japan , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Models, Molecular , Neurons/drug effects , Neurons/physiology , Protein Subunits/genetics , Protein Transport/genetics , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructure , Twins, Dizygotic
7.
Clin Calcium ; 24(2): 233-9, 2014 Feb.
Article in Japanese | MEDLINE | ID: mdl-24473356

ABSTRACT

There are four isozymes for human alkaline phosphatase (ALP) : tissue-nonspecific ALP (TNSALP), intestinal ALP, placental ALP and germ cell ALP. We present a brief history of TNSALP and review progress in research on it and a rare inborn error of metabolism called hypophosphatasia (HPP), which is caused by various loss-of-function mutations in the ALPL gene encoding TNSALP. HPP is characterized by decreased levels of serum ALP activity and defect in mineralization of bone and teeth, thus establishing the direct link between TNSALP and biomineralization. In addition to its 3D structure, studies on TNSALP mutants expressed in mammalian cells have revealed how each mutation affects the structure and function of TNSALP at the molecular level, which contributes to our understanding of the molecular basis of HPP.


Subject(s)
Alkaline Phosphatase/metabolism , Hypophosphatasia/enzymology , Hypophosphatasia/genetics , Isoenzymes/metabolism , Alkaline Phosphatase/genetics , Animals , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Genetic Predisposition to Disease , Genetic Testing/methods , Humans , Isoenzymes/genetics , Mutation/genetics
8.
Cytokine ; 64(3): 642-5, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24084331

ABSTRACT

IL-17RA, a member of the interleukin (IL)-17 receptor family, is a single membrane-spanning protein that ubiquitously expressed on the cell surface. IL-17RA transduces IL-17A, IL-17F, and IL-17A/F heterodimer-mediated signals by forming a complex with IL-17RC, and also signals the IL-17E (also known as IL-25) response in combination with IL-17RB (also known as IL-25R). Previously, soluble isoforms of human IL-17RC and IL-17RB have been reported, but the existence of a soluble isoform of human IL-17RA has remained unclear. Here, we report the identification of a soluble isoform of human IL-17RA at the mRNA and protein levels. Reverse transcribed PCR experiments showed that the IL-17RA variant is generated by spliced out of exon 11 encoding the transmembrane region in a variety of human tissues. The soluble IL-17RA isoform was detected in the culture media of human cell lines by Western blotting. The existence of the soluble IL-17RA isoform sheds new light on the regulation of IL-17RA mediated responses.


Subject(s)
Alternative Splicing , Exons/genetics , Gene Expression Profiling , Receptors, Interleukin-17/genetics , Base Sequence , Blotting, Western , Cell Line , Female , Humans , Male , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Interleukin-17/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Solubility
9.
Mol Biol Cell ; 24(18): 2907-17, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23885118

ABSTRACT

Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor-mediated initial contact followed by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum-localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to-trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.


Subject(s)
Cell Cycle Proteins/metabolism , Multiprotein Complexes/metabolism , SNARE Proteins/metabolism , trans-Golgi Network/metabolism , Cholera Toxin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Endoplasmic Reticulum/metabolism , Endosomes/metabolism , HeLa Cells , Humans , Microtubule-Associated Proteins/metabolism , Models, Biological , Protein Structure, Tertiary , Protein Transport , Syntaxin 16/metabolism
10.
Mol Genet Metab ; 109(3): 282-8, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23688511

ABSTRACT

Various loss-of function mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene cause a rare genetic disorder called hypophosphatasia (HPP), which is characterized by defective mineralization in the bones and teeth and a deficiency in serum alkaline phosphatase. A point mutation (c.1250A>G), which leads to replacement of an asparagine at position 417 of TNSALP with serine [TNSALP (N417S)], has been reported in a patient diagnosed with perinatal HPP (Sergi C. et al. Am, J. Med. Genet. 103, 235-240, 2001). In order to characterize the molecular properties of TNSALP (N417S), we expressed and analyzed TNSALP (N417S) both in COS-1 cells (transient expression) and CHO K1 Tet-On cells (inducible cell system). In contrast to wild-type TNSALP [TNSALP (W)], cells expressing TNSALP (N417S) lacked its alkaline phosphatase activity. However, this mutant underwent N-linked oligosaccharide processing and appeared on the cell surface similar to TNSALP (W). Importantly, this mutant failed to assemble into a dimer structure, which is needed for the catalytic function of TNSALP, as evidenced by newly developed SDS-PAGE as well as sucrose-density-gradient centrifugation. Substitution of the asparagine at position 417 with structurally related amino acids such as an aspartate and a glutamine also abolished the dimerization of TNSALP without perturbing its cell surface localization. Taken together, the asparagine at position 417 is crucial for the assembly and function of TNSALP, which may explain the severity of the N417S mutation.


Subject(s)
Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Amino Acid Substitution , Hypophosphatasia/genetics , Mutation , Alkaline Phosphatase/chemistry , Animals , Asparagine , CHO Cells , COS Cells , Chlorocebus aethiops , Codon , Cricetulus , Gene Expression , Humans , Hypophosphatasia/metabolism
11.
FEBS J ; 279(23): 4327-37, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23039266

ABSTRACT

Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene cause hypophosphatasia (HPP), an inborn error of metabolism characterized by defects in bone and teeth mineralization accompanying subnormal levels of serum alkaline phosphatase activity. Missense mutations at position 420 of TNSALP (standard nomenclature), which convert glycine to serine [TNSALP (G420S)] or alanine [TNSALP (G420A)], have been reported in perinatal and childhood HPP, respectively. When expressed in COS-1 cells, both TNSALP mutants were indistinguishable from wild-type TNSALP [TNSALP (W)] as evidenced by immunofluorescence and western blotting. Nevertheless, the two TNSALP mutants did not show substantial alkaline phosphatase activity. In agreement with transiently transfected cells, TNSALP (G420S) expressed in a Tet-On inducible expression system lacked its alkaline phosphatase activity, although this mutant was anchored to the cell surface lipid bilayers by glycosylphosphatidylinositol as an 80 kDa mature form bearing complex-type oligosaccharides like TNSALP (W). Importantly, TNSALP (G420S) was found to largely fail to assemble into the homodimer in contrast to TNSALP (W). Taken together, these results demonstrate that the glycine residue at position 420 is crucial for the subunit interaction of TNSALP and hence its catalytic function without affecting trafficking of monomeric TNSALP. We conclude that the dimerization defect is the molecular basis for perinatal HPP associated with the genotype G420S/G420S.


Subject(s)
Alkaline Phosphatase/chemistry , Alkaline Phosphatase/metabolism , Glycine/genetics , Hypophosphatasia/enzymology , Serine/genetics , Alkaline Phosphatase/genetics , Animals , Blotting, Western , CHO Cells , COS Cells , Centrifugation, Density Gradient , Chlorocebus aethiops , Cricetinae , Dimerization , Humans , Hypophosphatasia/genetics , Mutation/genetics
12.
Biochim Biophys Acta ; 1822(4): 581-8, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22266140

ABSTRACT

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-ß-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.


Subject(s)
Alkaline Phosphatase/metabolism , Disulfides/metabolism , Hypophosphatasia/genetics , Mutation , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Humans , Hypophosphatasia/metabolism , Ubiquitination
13.
Cell Struct Funct ; 36(2): 171-85, 2011.
Article in English | MEDLINE | ID: mdl-21757827

ABSTRACT

The Yip1 domain family (YIPF) proteins are homologues of yeast Yip1p and Yif1p, which are proposed to function in ER to Golgi transport. Here, we report the characterization of YIPF3 and YIPF4, homologues of human Yif1p and Yip1p, respectively. Immunofluorescence and immuno-electron microscopy showed that both YIPF3 and YIPF4 are clearly concentrated in the cis-Golgi. While YIPF4 was detected as a single mobility form consistent with its predicted molecular weight, three different mobility forms of YIPF3 were detected by western blotting. Biochemical and immunofluorescence experiments strongly indicated that YIPF3 is synthesized in the ER as a N-glycosylated form (40 kDa), is then O-glycosylated in the Golgi apparatus to become a lower mobility form (46 kDa) and finally becomes a higher mobility form cleaved at its C-terminal luminal domain (36 kDa). YIPF3 and YIPF4 form a complex in the Golgi apparatus, and this was suggested to be important for their proper localization and function. The knockdown of YIPF3 or YIPF4 in HeLa cells induced fragmentation of the Golgi apparatus, suggesting their involvement in the maintenance of the Golgi structure.


Subject(s)
Golgi Apparatus/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Glycosylation , Golgi Apparatus/chemistry , HeLa Cells , Humans , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
14.
Cell Struct Funct ; 36(1): 1-12, 2011.
Article in English | MEDLINE | ID: mdl-21150128

ABSTRACT

When increased production of secretory proteins overwhelms the capacity of the endoplasmic reticulum (ER) and the Golgi apparatus, eukaryotic cells expand their capacity to sustain secretory function. The capacity of the ER is enhanced by the mechanism called the ER stress response, but the mechanism regulating Golgi capacity (the Golgi stress response) has remained unclear. Here, we found that transcription of Golgi-related genes, including glycosylation enzymes as well as factors involved in post-Golgi vesicular transport and maintenance of Golgi structure, was upregulated upon treatment with monensin, an ionophore that disrupts the function of acidic organelles, including the Golgi apparatus and lysosomes by neutralizing their lumen. This transcriptional induction was found to be commonly regulated by a novel cis-acting element called the Golgi apparatus stress response element (GASE), whose consensus sequence is ACGTGgc. When the function of the Golgi apparatus was specifically disturbed by overexpression of GCP60, a Golgi-localized protein that binds to giantin, transcription from GASE was significantly induced. These results suggest that mammalian cells have the Golgi stress response, and that GASE regulates transcriptional induction involved in the Golgi stress response.


Subject(s)
Golgi Apparatus/physiology , Response Elements/genetics , Stress, Physiological/genetics , Transcriptional Activation/genetics , Base Sequence , Golgi Apparatus/drug effects , HeLa Cells , Humans , Monensin/pharmacology , Response Elements/drug effects , Stress, Physiological/drug effects , Transcriptional Activation/drug effects
15.
Traffic ; 11(12): 1552-66, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20874812

ABSTRACT

The coiled-coil Golgi membrane protein golgin-84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin-84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin-84 as the tether for COPI vesicles of intra-Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin-84 knockdown (KD) cells. The depletion of golgin-84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and cis-Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex-dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin-84. Surprisingly, the interaction between golgin-84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin-84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin-84 with COG plays an important role in the tethering process of intra-Golgi retrograde vesicle traffic.


Subject(s)
Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Biological Transport , Cell Line , Golgi Matrix Proteins , Humans , Hyaluronan Receptors , Lysosomal Membrane Proteins/metabolism , Protein Binding , SNARE Proteins/metabolism , Vesicular Transport Proteins/metabolism
16.
Clin Chim Acta ; 411(5-6): 359-63, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19968980

ABSTRACT

BACKGROUND: We recently found that an increased plasma concentration of protein-conjugated acrolein is a good biomarker for stroke. Therefore we determine whether the concentration of protein-conjugated acrolein is increased in saliva from patients with primary Sjögren's syndrome. METHODS: Stimulated whole-mixed saliva was collected from 10 patients and 13 control subjects. The concentration of protein-conjugated acrolein in saliva and plasma was measured by either Western blotting or enzyme-linked immunosorbent assay. RESULTS: The concentration of protein-conjugated acrolein, especially albumin-conjugated acrolein, was greatly increased in saliva from patients with primary Sjögren's syndrome (p<0.001). The concentration of protein-conjugated acrolein was inversely correlated with the flow rate of saliva. CONCLUSION: The results indicate that the concentration of protein-conjugated acrolein, a marker of cell or tissue damage, in saliva is well correlated with seriousness of primary Sjögren's syndrome.


Subject(s)
Acrolein/blood , Albumins/chemistry , Lysine/blood , Sjogren's Syndrome/blood , alpha-Amylases/blood , Acrolein/chemistry , Aged , Biomarkers/blood , Biomarkers/chemistry , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lysine/chemistry , Middle Aged , alpha-Amylases/chemistry , alpha-Amylases/metabolism
17.
Exp Cell Res ; 314(19): 3427-43, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18718466

ABSTRACT

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF4- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.


Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Endoplasmic Reticulum/ultrastructure , Fluorescent Antibody Technique , Golgi Apparatus/ultrastructure , HeLa Cells , Humans , Membrane Proteins/analysis , Membrane Proteins/genetics , Microscopy, Immunoelectron , Protein Transport , Vesicular Transport Proteins/analysis , Vesicular Transport Proteins/genetics
18.
FEBS J ; 275(11): 2727-37, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18422967

ABSTRACT

Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.


Subject(s)
Alanine/chemistry , Alkaline Phosphatase/chemistry , Hypophosphatasia/metabolism , Valine/chemistry , Alkaline Phosphatase/physiology , Animals , CHO Cells , COS Cells , Chlorocebus aethiops , Cricetinae , Cricetulus , Humans , Kinetics , Protein Conformation , Protein Folding , Protein Structure, Tertiary
19.
Traffic ; 8(3): 270-84, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17274799

ABSTRACT

The vesicle-tethering protein p115 functions in endoplasmic reticulum-Golgi trafficking. We explored the function of homologous region 2 (HR2) of the p115 head domain that is highly homologous with the yeast counterpart, Uso1p. By expression of p115 mutants in p115 knockdown (KD) cells, we found that deletion of HR2 caused an irregular assembly of the Golgi, which consisted of a cluster of mini-stacked Golgi fragments, and gathered around microtubule-organizing center in a microtubule-dependent manner. Protein interaction analyses revealed that p115 HR2 interacted with Cog2, a subunit of the conserved oligomeric Golgi (COG) complex that is known another putative cis-Golgi vesicle-tethering factor. The interaction between p115 and Cog2 was found to be essential for Golgi ribbon reformation after the disruption of the ribbon by p115 KD or brefeldin A treatment and recovery by re-expression of p115 or drug wash out, respectively. The interaction occurred only in interphase cells and not in mitotic cells. These results strongly suggested that p115 plays an important role in the biogenesis and maintenance of the Golgi by interacting with the COG complex on the cis-Golgi in vesicular trafficking.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Brefeldin A/pharmacology , Endoplasmic Reticulum/metabolism , Golgi Matrix Proteins , HeLa Cells , Humans , Membrane Proteins/genetics , Mutation , Protein Interaction Mapping , Protein Structure, Tertiary , Protein Synthesis Inhibitors/pharmacology , Protein Transport , SNARE Proteins/metabolism , Vesicular Transport Proteins/genetics
20.
Biochem Biophys Res Commun ; 338(2): 1268-74, 2005 Dec 16.
Article in English | MEDLINE | ID: mdl-16256943

ABSTRACT

Depletion of p115 with small interfering RNA caused fragmentation of the Golgi apparatus, resulting in dispersed distribution of stacked short cisternae and a vesicular structure (mini-stacked Golgi). The mini-stacked Golgi with cis- and trans-organization is functional in protein transport and glycosylation, although secretion is considerably retarded in p115 knockdown cells. The fragmented Golgi was further disrupted by treatment with breferdin A and reassembled into the mini-stacked Golgi by removal of the drug, as observed in control cells. In addition, p115 knockdown cells maintained retrograde transport from the Golgi to the endoplasmic reticulum, although the rate was not as efficient as in control cells. While no alternation of microtubule networks was found in p115 knockdown cells, the fragmented Golgi resembled those in cells treated with anti-microtubule drugs. The results suggest that p115 is involved in vesicular transport between endoplasmic reticulum and the Golgi, along with microtubule networks.


Subject(s)
Drosophila Proteins/deficiency , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Membrane Proteins/deficiency , Protein Transport/physiology , Gene Silencing , Golgi Matrix Proteins , HeLa Cells , Humans
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