Intracellular targeting of Cisd2/Miner1 to the endoplasmic reticulum

Abstract Background Cisd1 and Cisd2 proteins share very similar structures with an N-terminal membrane-anchoring domain and a C-terminal cytosolic domain containing an iron-cluster binding domain and ending with a C-terminal KKxx sequence. Despite sharing a similar structure, Cisd1 and Cisd2 are anchored to different compartments: mitochondria for Cisd1 and endoplasmic reticulum for Cisd2. The aim of this study was to identify the protein motifs targeting Cisd2 to the ER and ensuring its retention in this compartment. Results We used new recombinant antibodies to localize Cisd1 and Cisd2 proteins, as well as various protein chimeras. Cisd2 is targeted to the ER by its N-terminal sequence. It is then retained in the ER by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif, and of an ER-targeting transmembrane domain. As previously reported for Cisd1, Cisd2 can alter the morphology of the compartment in which it accumulates. Conclusion Although they share a very similar structure, Cisd1 and Cisd2 use largely different intracellular targeting motifs to reach their target compartment (mitochondria and endoplasmic reticulum, respectively).

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A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2

Journal of Virology ◽

10.1128/jvi.72.3.2183-2191.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2183-2191 ◽

Cited By ~ 166

Author(s):

Laurence Cocquerel ◽

Jean-Christophe Meunier ◽

André Pillez ◽

Czeslaw Wychowski ◽

Jean Dubuisson

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Transmembrane Domain ◽

Intracellular Localization ◽

Native Form ◽

Glycosyl Phosphatidylinositol ◽

Er Retention ◽

Er Targeting ◽

Necessary And Sufficient

ABSTRACT The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.

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Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling

BioMed Research International ◽

10.1155/2015/319454 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 8

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.

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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin Ii, on the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.150.4.719 ◽

2000 ◽

Vol 150 (4) ◽

pp. 719-730 ◽

Cited By ~ 39

Author(s):

Yuichiro Kida ◽

Masao Sakaguchi ◽

Mitsunori Fukuda ◽

Katsuhiko Mikoshiba ◽

Katsuyoshi Mihara

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Membrane ◽

Type I ◽

Hydrophobic Region ◽

Anchor Protein ◽

Nascent Polypeptide ◽

Charged Residues ◽

Er Targeting ◽

Signal Anchor ◽

Positively Charged

Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

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Interaction of the endoplasmic reticulum α1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system

Journal of Cell Science ◽

10.1242/jcs.114.24.4629 ◽

2001 ◽

Vol 114 (24) ◽

pp. 4629-4635

Author(s):

Michel J. Massaad ◽

Annette Herscovics

Keyword(s):

Endoplasmic Reticulum ◽

Catalytic Domain ◽

Transmembrane Domain ◽

Protein A ◽

Yeast Cells ◽

Type Ii ◽

Ubiquitin System ◽

Endoplasmic Reticulum Protein ◽

Split Ubiquitin

The α1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi α1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Δpep4 cells and in the vacuoles of rer1/Δpep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of β-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.

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Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high- affinity IL-3 receptor

Blood ◽

10.1182/blood.v79.4.895.895 ◽

1992 ◽

Vol 79 (4) ◽

pp. 895-903

Author(s):

T Ogorochi ◽

T Hara ◽

HM Wang ◽

K Maruyama ◽

A Miyajima

Keyword(s):

Monoclonal Antibodies ◽

Transmembrane Domain ◽

Signal Sequence ◽

Amino Acid Level ◽

Terminal Sequence ◽

Chimeric Receptors ◽

Interleukin 3 ◽

High Affinity ◽

Processing Site ◽

Binding Component

Abstract Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody- producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL- 3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.

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Analysis of Structural Signals Conferring Localisation of Pig OST48 to the Endoplasmic Reticulum

Biological Chemistry ◽

10.1515/bc.2001.130 ◽

2001 ◽

Vol 382 (7) ◽

pp. 1039-1047 ◽

Cited By ~ 4

Author(s):

Birgit Hardt ◽

Raquel Aparicio ◽

Wilhelm Breuer ◽

Ernst Bause

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Transmembrane Domain ◽

Membrane Topology ◽

Type I ◽

Catalytic Subunits ◽

Hybrid Proteins ◽

Lysine Residues ◽

Typical Type ◽

Hydrophobic Transmembrane Domain

Abstract Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the cotranslational transfer of GlcNAc[2]Man[9]Glc[3] from Dol PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ERtargeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man[9] mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ERresidency on OST48. The doublelysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48?K-3, when coexpressed in COS-1 cells together with myctagged ribophorin I, was quantitatively retained in the ER. By contrast, coexpression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMO?K-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the Cterminus of the Man[9]mannosidase luminal domain. Thus ERlocalisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the doublelysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in recapture of molecules which have escaped from the ER.

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