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.

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 Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Monica Giannotta ◽  
Giorgia Fragassi ◽  
Antonio Tamburro ◽  
Capone Vanessa ◽  
Alberto Luini ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Transmembrane Domain ◽  
Cell Cycle Control ◽  
Retrograde Transport ◽  
Multifunctional Protein ◽  
G Protein Coupled ◽  
Kdel Receptor ◽  
Prohibitin 1

The KDEL receptor (KDELR) is a seven-transmembrane-domain protein involved in retrograde transport of protein chaperones from the Golgi complex to the endoplasmic reticulum. Our recent findings have shown that the Golgi-localised KDELR acts as a functional G-protein-coupled receptor by binding to and activating Gs and Gq. These G proteins induce activation of PKA and Src and regulate retrograde and anterograde Golgi trafficking. Here we used an integrated coimmunoprecipitation and mass spectrometry approach to identify prohibitin-1 (PHB) as a KDELR interactor. PHB is a multifunctional protein that is involved in signal transduction, cell-cycle control, and stabilisation of mitochondrial proteins. We provide evidence that depletion of PHB induces intense membrane-trafficking activity at the ER–Golgi interface, as revealed by formation of GM130-positive Golgi tubules, and recruitment of p115,β-COP, and GBF1 to the Golgi complex. There is also massive recruitment of SEC31 to endoplasmic-reticulum exit sites. Furthermore, absence of PHB decreases the levels of the Golgi-localised KDELR, thus preventing KDELR-dependent activation of Golgi-Src and inhibiting Golgi-to-plasma-membrane transport of VSVG. We propose a model whereby in analogy to previous findings (e.g., the RAS-RAF signalling pathway), PHB can act as a signalling scaffold protein to assist in KDELR-dependent Src activation.

The open reading frame 1-encoded (‘36K’) protein of Carnation Italian ringspot virus localizes to mitochondria

2001 ◽  
Vol 82 (1) ◽  
pp. 29-34 ◽  
Author(s):  
L. Rubino ◽  
F. Weber-Lotfi ◽  
A. Dietrich ◽  
C. Stussi-Garaud ◽  
M. Russo
Keyword(s):  
Outer Membrane ◽  
Fluorescent Protein ◽  
Amorphous Material ◽  
Subcellular Fractionation ◽  
Open Reading Frame ◽  
Ringspot Virus ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Reading Frame ◽  
Open Reading Frame 1

The localization of the 36 kDa (‘36K’) protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K–GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

Recycling of the yeast v-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec20p

1998 ◽  
Vol 111 (11) ◽  
pp. 1507-1520 ◽  
Author(s):  
W. Ballensiefen ◽  
D. Ossipov ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Hybrid Protein ◽  
Wild Type ◽  
Alpha Factor ◽  
Membrane Compartments

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

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 Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

1995 ◽  
Vol 6 (11) ◽  
pp. 1459-1477 ◽  
Author(s):  
K Sato ◽  
S Nishikawa ◽  
A Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Subcellular Fractionation ◽  
Restrictive Temperature ◽  
Functional Protein ◽  
Reading Frame ◽  
C Terminus ◽  
Retention System ◽  
Punctate Pattern ◽  
Membrane Spanning

Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyses have suggested that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early region of the Golgi apparatus. The rer1 mutant we isolated in a previous study mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene and determined its reading frame. RER1 encodes a hydrophobic protein of 188 amino acid residues containing four putative membrane spanning domains. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an epitope derived from the influenza hemagglutinin was added to the C-terminus of Rer1p and the cells expressing this tagged but functional protein were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern that is typical for Golgi proteins and clearly distinct from the ER staining. This punctate staining was in fact exaggerated in the sec7 mutant that accumulates the Golgi membranes at the restrictive temperature. Furthermore, double staining of Rer1p and Ypt1p, a GTPase that is known to reside in the Golgi apparatus, showed good colocalization. Subcellular fractionation experiments indicated that the fractionation pattern of Rer1p was similar to that of an early Golgi protein, Och1p. From these results, we suggest that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.

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 Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

1996 ◽  
Vol 133 (4) ◽  
pp. 777-789 ◽  
Author(s):  
I V Majoul ◽  
P I Bastiaens ◽  
H D Söling
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Retrograde Transport ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Pulse Treatment ◽  
Resistant Form ◽  
Immunofluorescence Studies ◽  
Intermediate Compartment ◽  
Kdel Sequence

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.

scholarly journals An Engineered Glutamate-gated Chloride (GluCl) Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin

2013 ◽  
Vol 288 (29) ◽  
pp. 21029-21042 ◽  
Author(s):  
Shawnalea J. Frazier ◽  
Bruce N. Cohen ◽  
Henry A. Lester
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Neuronal Cultures ◽  
Β Subunit ◽  
Intracellular Loop ◽  
High Concentration ◽  
Α Subunit ◽  
Endoplasmic Reticulum Retention ◽  
Channel Effects

A modified invertebrate glutamate-gated Cl− channel (GluCl αβ) was previously employed to allow pharmacologically induced silencing of electrical activity in CNS neurons upon exposure to the anthelmintic drug ivermectin (IVM). Usefulness of the previous receptor was limited by 1) the high concentration of IVM necessary to elicit a consistent silencing phenotype, raising concern about potential side effects, and 2) the variable extent of neuronal spike suppression, due to variations in the co-expression levels of the fluorescent protein-tagged α and β subunits. To address these issues, mutant receptors generated via rational protein engineering strategies were examined for improvement. Introduction of a gain-of-function mutation (L9′F) in the second transmembrane domain of the α subunit appears to facilitate β subunit incorporation and substantially increase heteromeric GluCl αβ sensitivity to IVM. Removal of an arginine-based endoplasmic reticulum retention motif (RSR mutated to AAA) from the intracellular loop of the β subunit further promotes heteromeric expression at the plasma membrane possibly by preventing endoplasmic reticulum-associated degradation of the β subunit rather than simply reducing endoplasmic reticulum retention. A monomeric XFP (mXFP) mutation that prevents fluorescent protein dimerization complements the mutant channel effects. Expression of the newly engineered GluCl opt α-mXFP L9′F + opt β-mXFP Y182F RSR_AAA receptor in dissociated neuronal cultures markedly increases conductance and reduces variability in spike suppression at 1 nm IVM. This receptor, named “GluClv2.0,” is an improved tool for IVM-induced silencing.

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