scholarly journals Targeting of inositol 1,4,5-trisphosphate receptor to the endoplasmic reticulum by its first transmembrane domain

2009 ◽  
Vol 425 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Evangelia Pantazaka ◽  
Colin W. Taylor
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Subcellular Fractionation ◽  
Hydrophobic Residues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Stable Association ◽  
Green Fluorescent ◽  
Trisphosphate Receptor

Targeting of IP3R (inositol 1,4,5-trisphosphate receptors) to membranes of the ER (endoplasmic reticulum) and their retention within ER or trafficking to other membranes underlies their ability to generate spatially organized Ca2+ signals. N-terminal fragments of IP3R1 (type 1 IP3R) were tagged with enhanced green fluorescent protein, expressed in COS-7 cells and their distribution was determined by confocal microscopy and subcellular fractionation. Localization of IP3R1 in the ER requires translation of between 26 and 34 residues beyond the end of the first transmembrane domain (TMD1), a region that includes TMD2 (second transmembrane domain). Replacement of these post-TMD1 residues with unrelated sequences of similar length (24–36 residues) partially mimicked the native residues. We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER. Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

scholarly journals Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (7) ◽  
pp. 2518-2532 ◽  
Author(s):  
Ken Sato ◽  
Akihiko Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Terminal Region ◽  
Growth Defect ◽  
Critical Determinant ◽  
Reading Frame ◽  
Glycoprotein Secretion

The yeast open reading frame YLR080w/EMP46 encodes a homolog of the Golgi protein Emp47p. These two proteins are 45% identical and have a single transmembrane domain in their C-terminal regions and a carbohydrate recognition domain signature in the N-terminal region. The C-terminal tail of Emp46p includes a dilysine signal. This protein is localized to Golgi membranes at steady state by subcellular fractionation and green fluorescent protein labeling. On block of forward transport in sec12-4 cells, redistribution of Emp46p from the Golgi to the endoplasmic reticulum is observed. These localization features are similar to those previously reported for Emp47p. In addition, mutagenesis of the C-terminal region identified a tyrosine-containing motif as a critical determinant of the Golgi-localization and interaction with both COPI and COPII components. Similar motifs are also observed in the C-terminal tail of Emp47p and other mammalian homologs. Disruption of Emp47p displays a growth defect at a high temperature or on Ca2+-containing medium, which is rescued by overexpression of Emp46p, suggesting a partially overlapping function between Emp46p and Emp47p. In addition, we found that the disruption of both Emp46p and Emp47p show a marked defect in the secretion of a subset of glycoproteins. Analysis of the C-terminal mutants for Ca2+ sensitivity revealed that the forward transport of Emp46/47p is essential for their function, whereas the retrograde transport is not. We propose that Emp46p and Emp47p are required for the export of specific glycoprotein cargo from the endoplasmic reticulum.

Human GLUD1 and GLUD2 glutamate dehydrogenase localize to mitochondria and endoplasmic reticulum

2009 ◽  
Vol 87 (3) ◽  
pp. 505-516 ◽  
Author(s):  
Vasileios Mastorodemos ◽  
Dimitra Kotzamani ◽  
Ioannis Zaganas ◽  
Giovanna Arianoglou ◽  
Helen Latsoudis ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Glutamate Dehydrogenase ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Expression Vectors ◽  
Subcellular Targeting ◽  
Western Blots ◽  
Green Fluorescent ◽  
Coarse Structures ◽  
Egfp Fluorescence

Mammalian glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is thought to localize to the mitochondrial matrix, although there are also suggestions for the extramitochondrial presence of this protein. Whereas GDH in mammals is encoded by the GLUD1 gene, humans and the great apes have, in addition, a GLUD2 gene showing a distinct expression pattern. The encoded hGDH1 and hGDH2 isoenzymes are highly homologous, but their leader sequences are more divergent. To explore their subcellular targeting, we constructed expression vectors in which hGDH1 or hGDH2 was fused with the enhanced green fluorescent protein (EGFP) and used these to transfect COS 7, HeLa, CHO, HEK293, or neuroblastoma SHSY-5Y cells. Confocal microscopy revealed GDH-EGFP fluorescence in the cytoplasm within coarse structures. Cotransfection experiments using organelle-specific markers revealed that hGDH1 or hGDH2 colocalized with the mitochondrial marker DsRed2-Mito and to a lesser extent with the endoplasmic reticulum marker DsRed2-ER. Western blots detected two GDH-EGFP specific bands: a ~90 kDa band and a ~95 kDa band associated with the mitochondria and the endoplasmic reticulum containing cytosol, respectively. Deletion of the signal sequence, while altering drastically the fluoresce distribution within the cell, prevented GDH from entering the mitochondria, with the ~90 kDa band being retained in the cytosol. In addition, the deletion eliminated the ~95 kDa band from cell lysates, thus confirming that it represents the full-length GDH. Hence, while most of the hGDHs translocate into the mitochondria (a process associated with cleavage of the signal sequence), part of the protein localizes to the endoplasmic reticulum, probably serving additional functions.

scholarly journals Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

2001 ◽  
Vol 12 (7) ◽  
pp. 2031-2046 ◽  
Author(s):  
Nina Tolonen ◽  
Laura Doglio ◽  
Sibylle Schleich ◽  
Jacomine Krijnse Locker
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Nuclear Envelope Assembly ◽  
Green Fluorescent Protein Tagging ◽  
Green Fluorescent

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

scholarly journals Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

2001 ◽  
Vol 152 (5) ◽  
pp. 935-944 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Akihiko Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Endoplasmic Reticulum Membrane ◽  
Golgi Membrane ◽  
Green Fluorescent

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

scholarly journals Regulated Degradation of an Endoplasmic Reticulum Membrane Protein in a Tubular Lysosome in Leishmania mexicana

2001 ◽  
Vol 12 (8) ◽  
pp. 2364-2377 ◽  
Author(s):  
Kylie A. Mullin ◽  
Bernardo J. Foth ◽  
Steven C. Ilgoutz ◽  
Judy M. Callaghan ◽  
Jody L. Zawadzki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stationary Phase ◽  
Serine Proteases ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Leishmania Mexicana ◽  
Key Enzyme ◽  
Green Fluorescent ◽  
Major Surface

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.

scholarly journals Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor–green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation

1997 ◽  
Vol 323 (1) ◽  
pp. 273-280 ◽  
Author(s):  
Lee G. SAYERS ◽  
Atsushi MIYAWAKI ◽  
Akira MUTO ◽  
Hiroshi TAKESHITA ◽  
Akitsugu YAMAMOTO ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Animal Pole ◽  
Inositol Trisphosphate Receptor ◽  
Intracellular Targeting ◽  
Fluorescence Confocal Microscopy ◽  
Inositol 1,4,5 Trisphosphate ◽  
Microscopy Data ◽  
Green Fluorescent ◽  
Trisphosphate Receptor

In an attempt to define structural regions of the type I inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4,5)P3R(ES) fusion protein in Xenopus laevisoocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4,5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4,5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4,5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

scholarly journals The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

2007 ◽  
Vol 82 (5) ◽  
pp. 2161-2169 ◽  
Author(s):  
Amalia K. Earley ◽  
Winnie M. Chan ◽  
Brian M. Ward
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Gfp Fluorescence ◽  
Virion Morphogenesis ◽  
Green Fluorescent

ABSTRACT The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/ΔA34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/ΔA34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.

scholarly journals Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca2+ release and entry in DT40 B lymphocytes

2004 ◽  
Vol 382 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Takao MORITA ◽  
Akihiko TANIMURA ◽  
Akihiro NEZU ◽  
Tomohiro KUROSAKI ◽  
Yosuke TOJYO
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Receptor ◽  
Receptor Type ◽  
Intact Cells ◽  
Entry Pathway ◽  
Inositol 1,4,5 Trisphosphate ◽  
Green Fluorescent ◽  
Trisphosphate Receptor ◽  
Type 3

We examined the function of GFP-IP3R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca2+ release and entry using a mutant DT40 cell line (IP3R-KO) in which all three IP3R genes had been disrupted. GFP-IP3R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP3R3 apparently co-localized with the plasma membrane. The application of IP3 to permeabilized WT (wild-type) DT40 cells induced Ca2+ release from internal stores. Although this did not occur in IP3R-KO cells it was restored by expression of GFP-IP3R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca2+ response in IP3R-KO cells, whereas these treatments induced oscillatory or transient Ca2+ responses in GFP-IP3R3-expressing IP3R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca2+ entry in WT and GFP-IP3R3-expressing IP3R-KO cells but not in IP3R-KO cells. This BCR-mediated Ca2+ entry was observed in the presence of La3+, which blocks capacitative Ca2+ entry. Thapsigargin depleted Ca2+ stores and led to Ca2+ entry in IP3R-KO cells irrespective of GFP-IP3R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca2+ entry was completely blocked by La3+, suggesting that the BCR-mediated Ca2+ entry pathway is distinct from the capacitative Ca2+ entry pathway. The present study demonstrates that GFP-IP3R3 could compensate for native IP3R in both IP3-induced Ca2+ release and BCR-mediated Ca2+ entry.

scholarly journals Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways

2001 ◽  
Vol 12 (8) ◽  
pp. 2482-2496 ◽  
Author(s):  
Nica Borgese ◽  
Ilaria Gazzoni ◽  
Massimo Barberi ◽  
Sara Colombo ◽  
Emanuela Pedrazzini
Keyword(s):  
Endoplasmic Reticulum ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mitochondrial Outer Membrane ◽  
C Terminus ◽  
Green Fluorescent ◽  
Ta Proteins

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b 5, a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.

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