Biomedical subjects

Masatoshi Maki

Publications and source records attributed to Masatoshi Maki.

At least 19 recordsLinked to original sources

PubMed2006

ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner.

Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport.

Animals↗

PubMed2006

HD-PTP and Alix share some membrane-traffic related proteins that interact with their Bro1 domains or proline-rich regions.

Mammalian Alix is a multifunctional adaptor protein involved in cell death, receptor endocytosis, endosomal protein sorting and cell adhesion by associating with various proteins such as ALG-2, CIN85/Rukl/SETA, endophilins, CHMP4s and TSG101. HD-PTP is a paralog of Alix and a putative protein tyrosine phosphatase (PTP) that contains a Bro1 domain, coiled-coils, a proline-rich region (PRR) in addition to a PTP domain. We investigated interactions between HD-PTP and Alix-binding proteins. In the yeast two-hybrid assay, HD-PTP showed positive interactions with CHMP4b/Shax1, TSG101, endophilin A1 and ALG-2 but not with either RabGAPLP or CIN85. We confirmed the interactions in a mammalian system by Strep-pulldown assays in which pulldown products from the lysates of HEK293T cells expressing either Strep-tagged HD-PTP alone or co-expressing with epitope-tagged proteins were analyzed by Western blotting using specific antibodies. While Alix associated with both ALG-2 and TSG101 in a Ca2+-dependent manner, HD-PTP interacted with ALG-2 Ca2+-dependently but with TSG101 Ca2+-independently.

Adaptor Proteins, Signal Transducing↗

PubMed2006

CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway.

All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1-CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG-CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG-CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b-GFP or GFP-Vps4B(E235Q), a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP-CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP-CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.

Amino Acid Sequence↗

PubMed2006

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels.

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.

Amino Acid Motifs↗

PubMed2006

Calpain system regulates the differentiation of adult primitive mesenchymal ST-13 adipocytes.

The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-alpha (C/EBPalpha), peroxisome proliferator-activated receptor (PPAR)-gamma, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor alpha, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca(2+)-dependent degradation of PPARgamma and C/EBPalpha but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARgamma and C/EBPalpha. Under both conditions, calpain inhibitors almost completely prevented C/EBPalpha cleavage but partially blocked the decrease of PPARgamma. Two ubiquitous forms of calpain, mu- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of mu- but not m-calpain was found in the nucleus. Finally, stable dominant-negative mu-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARgamma and C/EBPalpha during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.

Adipocytes↗

PubMed2006

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA.

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

Amino Acid Sequence↗

PubMed2005

The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B.

ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 did not co-localize with GFP-SKD1(E235Q). Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Overexpression of calpastatin inhibits L8 myoblast fusion.

The formation of skeletal muscle fibers involves cessation of myoblast division, myoblast alignment, and fusion to multinucleated myofibers. Calpain is one of the factors shown to be involved in myoblast fusion. Using L8 rat myoblasts, we found that calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished prior to myoblast fusion and reappeared after fusion. The transient diminution in calpastatin allows the Ca2+-promoted activation of calpain and calpain-induced membrane proteolysis, which is required for myoblast fusion. Here we show that calpastatin overexpression in L8 myoblasts does not inhibit cell proliferation and alignment, but prevents myoblast fusion and fusion-associated protein degradation. In addition, calpastatin appears to modulate myogenic gene expression, as indicated by the lack of myogenin (a transcription factor expressed in differentiating myoblasts) in myoblasts overexpressing calpastatin. These results suggest that, in addition to the role in membrane disorganization in the fusing myoblasts, the calpain-calpastatin system may also modulate the levels of factors required for myoblast differentiation.

Animals↗

PubMed2005

Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting.

CHMP6 (charged multivesicular body protein 6) is a human orthologue of yeast Vps (vacuolar protein sorting) 20, a component of ESCRT (endosomal sorting complex required for transport)-III. Various CHMP6 orthologues in organisms ranging from yeast to humans contain the N-myristoylation consensus sequence at each N-terminus. Metabolic labelling of HEK-293 (human embryonic kidney) cells showed the incorporation of [3H]myristate into CHMP6 fused C-terminally to GFP (green fluorescent protein) (CHMP6-GFP). Interactions of CHMP6 with another ESCRT-III component CHMP4b/Shax [Snf7 (sucrose non-fermenting 7) homologue associated with Alix] 1, one of three paralogues of human Vps32/Snf7, and with EAP20 (ELL-associated protein 20), a human counterpart of yeast Vps25 and component of ESCRT-II, were observed by co-immunoprecipitation of epitope-tagged proteins expressed in HEK-293 cells. The in vitro pull-down assays using their recombinant proteins purified from Escherichia coli demonstrated direct physical interactions which were mediated by the N-terminal basic half of CHMP6. Overexpressed CHMP6-GFP in HeLa cells exhibited a punctate distribution throughout the cytoplasm especially in the perinuclear area, as revealed by fluorescence microscopic analysis. Accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of an MVB (multivesicular body), was observed in the CHMP6-GFP-localizing area. FLAG-tagged EAP20 distributed diffusely, but exhibited a punctate distribution on co-expression with CHMP6-GFP. Overexpression of CHMP6-GFP caused reduction of transferrin receptors on the plasma membrane surface, but caused their accumulation in the cytoplasm. Ubiquitinated proteins and endocytosed EGF continuously accumulated in CHMP6-GFP-expressing cells. These results suggest that CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes and regulates cargo sorting.

Amino Acid Sequence↗

PubMed2005

Dictyostelium discoideum requires an Alix/AIP1 homolog, DdAlix, for morphogenesis in alkaline environments.

Alix and its homologs are involved in various phenomena such as endosomal protein-sorting and adaptation to stress conditions. In this study, we found that development of Dictyostelium discoideum Alix (DdAlix) deletion mutant (alx-) cells was impaired in alkaline pH environments. The fruiting body formation efficiency of alx- cells at pH 9.0 was significantly lower than that of wild-type cells (6.8+/-4.2% vs 93+/-6.3%). The alkaline-sensitive phenotype of alx- cells was rescued by addition of salt. The phenotype was rescued by exogenous expression of human Alix as well as DdAlix but not by that of either Saccharomyces cerevisiae Alix homolog Rim20 or Bro1. DdAlix may be, structurally and functionally, more related to human Alix than to yeast Rim20 and Bro1.

Alkalies↗

PubMed2005

Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors.

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.

Animals↗

PubMed2005

Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice.

Calpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases.

Animals↗

PubMed2005

Reevaluation of the predicted gene structure of Dictyostelium cystatin A3 (cpiC) by nucleotide sequence determination of its cDNA* and its phylogenetic position in the cystatin superfamily.

Cystatins, cysteine protease inhibitors, are widely distributed among eukaryotes. We reevaluated the structure of the gene cpiC, a gene encoding the third identified member of cystatin family (cystatin A3) that was predicted in the genome database of the social amoeba Dictyostelium discoidium (dictyBase) but remained controversial. We determined the sequences of cDNA and PCR-amplified genomic DNA fragment and found a critical error in the registered nucleotide sequence. The corrected cystatin A3 gene has an open reading frame (ORF) without intron sequence interruption and encodes 94 amino acids (aa), in contrast to the previously predicted sequence of either 80, 82 or 118 aa. The cDNA has an unusual internal poly(A) sequence of 31 adenines, which immediately follows the translation termination codon (TAA) located 146 nucleotides upstream of the post-transcriptional polyadenylation site. The amino acid sequence of Dictyostelium cystatin A3 shows a high similarity to those of previously reported Dictyostelium cystatins as well as Family I cystatins of higher eukaryotes.

Amino Acid Sequence↗

PubMed2005

Identification of Rab GTPase-activating protein-like protein (RabGAPLP) as a novel Alix/AIP1-interacting protein.

Alix/AIP1 is a multifunctional adaptor protein involved in endocytosis, cell adhesion, and cell death. By yeast two-hybrid screening we identified a novel Alix/AIP1-interacting protein named Rab GTPase-activating protein-like protein (RabGAPLP). Interaction between Alix and RabGAPLP was confirmed by pull-down assays using fusion proteins of either glutathione-S-transferase (GST) or chitin-binding domain (CBD) and lysates of cultured mammalian cells expressing the respective proteins. Partial colocalization of FLAG-tagged RabGAPLP and green fluorescent protein (GFP)-fused Alix was observed at cell edges and filopodia-like structures by fluorescence confocal laser scanning microscopic analysis. The identity of RabGAPLP to merlin-associated protein (MAP), one of the interacting partners of neurofibromatosis type 2 (NF2) tumor suppressor gene product (merlin), implies cross-talk of membrane traffic and cell adhesion.

Calcium-Binding Proteins↗

PubMed2004

DdAlix, an Alix/AIP1 homolog in Dictyostelium discoideum, is required for multicellular development under low Ca2+ conditions.

Apoptosis-linked gene 2 (ALG-2) interacting protein X (Alix), also called AIP1, is a widely conserved protein in eukaryotes. Alix and its homologs are involved in various phenomena such as apoptosis, regulation of cell adhesion, protein sorting, adaptation to stress conditions, and budding of human immunodeficiency virus (HIV). To investigate the role of Alix in development, we identified an Alix homolog in the cellular slime mold Dictyostelium discoideum and disrupted the gene by homologous recombination. The growth of DdAlix deletion mutant (alx-) cells was significantly impaired in the presence of 5 mM Li+. On an agar plate, alx- cells underwent normal development and formed fruiting bodies indistinguishable from those formed by wild-type cells. However, alx- cells could not form fruiting bodies in the presence of 5 mM Li+. Similar results were obtained when cells were developed in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), which is an antagonist of intracellular Ca2+ store. Furthermore, when the extracellular free Ca2+ was reduced to 10 nM, the ability of alx- cells, but not that of wild-type cells, to form fruiting bodies was impaired. The results indicate that DdAlix is essential for development under low Ca2+ conditions and suggest that DdAlix is involved in Ca2+ signaling during development.

Animals↗

PubMed2004

CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms.

The ALG-2-interacting protein Alix has recently been demonstrated to associate with CHMP4b that is a human homologue of yeast Snf7p (also named Vps32p) and a member of the family of small coiled-coil proteins named CHMP implicated in playing roles in multivesicular body sorting. In addition to the previously isolated cDNAs for two CHMP4 proteins (CHMP4a and CHMP4b), we isolated a cDNA for a new member of the CHMP4 subfamily (designated CHMP4c). Northern blot analyses revealed different expression patterns of the mRNAs for the three CHMP4 isoforms in human tissues. CHMP4b messages were expressed at higher levels in all 12 tissues tested in comparison with the CHMP4a and CHMP4c transcripts, particularly in heart and skeletal muscle. The interaction with Alix was detected for each CHMP4 isoform by co-immunoprecipitation experiments using lysates of HEK293 cells expressing each epitope-tagged CHMP4 protein and Alix fused with green fluorescent protein. Further, using recombinant glutathione S-transferase (GST) fusion protein of truncated Alix (amino acids 1-423) and thioredoxin-tagged CHMP4 proteins, the direct interactions were detected by a GST pull-down assay, where CHMP4b showed a stronger interaction than other CHMP4 isoforms. These results suggest that CHMP4b is a major binding partner of Alix among the three CHMP4 isoforms.

Amino Acid Sequence↗

PubMed2004

The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1.

ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca(2+)-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca(2+) with FLAG-tagged Alix or FLAG-tagged Alix Delta EBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged Alix Delta ABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca(2+)-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca(2+)-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca(2+). Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCT Delta ABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca(2+)-dependent interaction with Alix.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Analyses of expression and localization of two mammalian-type transglutaminases in Physarum polycephalum, an acellular slime mold.

Transglutaminase (TGase) is an enzyme that modifies proteins by crosslinking or polyamination. Physarum polycephalum, an acellular slime mold, is the evolutionally lowest organism that has a mammalian-type transglutaminase. We have cloned a cDNA for Physarum polycephalum TGase (PpTGB), homologous to a previously identified TGase (PpTGA), whose sequence is similar to that of mammalian TGases. PpTGB encodes a primary sequence identical to that of PpTGA except for 11 amino acid residues at the N-terminus. Reverse transcription-PCR and Western blotting analyses showed that both PpTGA and PpTGB are expressed in microplasmodia and macroplasmodia during their life cycle, except for in sporangia. For biochemical characterization, we carried out the ectopical expressions of PpTGA and PpTGB in Dictyostelium discoideum. Subcellular fractionation of these Dictyostelium cells showed that the expressed PpTGA, but not PpTGB, localizes to the membrane fraction. Furthermore, in Physarum, subcellular fractionation and immunostaining indicated specific localization at the plasma membrane in macroplasmodia, while the localization was entirely cytoplasmic in microplasmodia.

Amino Acid Sequence↗