scholarly journals Endoplasmic Reticulum Export of Glycosyltransferases Depends on Interaction of a Cytoplasmic Dibasic Motif with Sar1

2003 ◽  
Vol 14 (9) ◽  
pp. 3753-3766 ◽  
Author(s):  
Claudio G. Giraudo ◽  
Hugo J.F. Maccioni
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Type I ◽  
Type Ii ◽  
Transport Vesicles ◽  
Microsomal Membranes ◽  
Copii Vesicles ◽  
Er Export ◽  
Selective Process ◽  
Selection Of

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p–Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

scholarly journals Erv14p Directs a Transmembrane Secretory Protein into COPII-coated Transport Vesicles

2002 ◽  
Vol 13 (3) ◽  
pp. 880-891 ◽  
Author(s):  
Jacqueline Powers ◽  
Charles Barlowe
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Conserved Residues ◽  
Early Secretory Pathway ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Selection Of

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.

Labelling of non-A, non-B hepatitis infected chimpanzee hepatocytes with nuclease-gold conjugates

1984 ◽  
Vol 42 ◽  
pp. 250-251
Author(s):  
G. D. Gagne ◽  
M. F. Miller ◽  
D. A. Peterson
Keyword(s):  
Endoplasmic Reticulum ◽  
Experimental Infection ◽  
Uranyl Acetate ◽  
Type I ◽  
Cellular Injury ◽  
Type Ii ◽  
Tubular Structures ◽  
Type Iii ◽  
Type Iv ◽  
Infected Chimpanzee

Experimental infection of chimpanzees with non-A, non-B hepatitis (NANB) or with delta agent hepatitis results in the appearance of characteristic cytoplasmic alterations in the hepatocytes. These alterations include spongelike inclusions (Type I), attached convoluted membranes (Type II), tubular structures (Type III), and microtubular aggregates (Type IV) (Fig. 1). Type I, II and III structures are, by association, believed to be derived from endoplasmic reticulum and may be morphogenetically related. Type IV structures are generally observed free in the cytoplasm but sometimes in the vicinity of type III structures. It is not known whether these structures are somehow involved in the replication and/or assembly of the putative NANB virus or whether they are simply nonspecific responses to cellular injury. When treated with uranyl acetate, type I, II and III structures stain intensely as if they might contain nucleic acids. If these structures do correspond to intermediates in the replication of a virus, one might expect them to contain DNA or RNA and the present study was undertaken to explore this possibility.

scholarly journals The Type II Deiodinase Is Retrotranslocated to the Cytoplasm and Proteasomes via p97/Atx3 Complex

2013 ◽  
Vol 27 (12) ◽  
pp. 2105-2115 ◽  
Author(s):  
Rafael Arrojo e Drigo ◽  
Péter Egri ◽  
Sungro Jo ◽  
Balázs Gereben ◽  
Antonio C. Bianco
Keyword(s):  
Molecular Weight ◽  
Endoplasmic Reticulum ◽  
Thyroid Hormone ◽  
High Molecular Weight ◽  
20S Proteasome ◽  
Type I ◽  
Type Ii ◽  
Cell Models ◽  
Natural Substrate ◽  
Iodothyronine Deiodinase

The type II iodothyronine deiodinase (D2) is a type I endoplasmic reticulum (ER)-resident thioredoxin fold-containing selenoprotein that activates thyroid hormone. D2 is inactivated by ER-associated ubiquitination and can be reactivated by two ubiquitin-specific peptidase-class D2-interacting deubiquitinases (DUBs). Here, we used D2-expressing cell models to define that D2 ubiquitination (UbD2) occurs via K48-linked ubiquitin chains and that exposure to its natural substrate, T4, accelerates UbD2 formation and retrotranslocation to the cytoplasm via interaction with the p97-ATPase complex. D2 retrotranslocation also includes deubiquitination by the p97-associated DUB Ataxin-3 (Atx3). Inhibiting Atx3 with eeyarestatin-I did not affect D2:p97 binding but decreased UbD2 retrotranslocation and caused ER accumulation of high-molecular weight UbD2 bands possibly by interfering with the D2-ubiquitin-specific peptidases binding. Once in the cytosol, D2 is delivered to the proteasomes as evidenced by coprecipitation with 19S proteasome subunit S5a and increased colocalization with the 20S proteasome. We conclude that interaction between UbD2 and p97/Atx3 mediates retranslocation of UbD2 to the cytoplasm for terminal degradation in the proteasomes, a pathway that is accelerated by exposure to T4.

scholarly journals Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (9) ◽  
pp. 2681-2697 ◽  
Author(s):  
Kenneth Moss ◽  
Andrew Helm ◽  
Yun Lu ◽  
Alvina Bragin ◽  
William R. Skach
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Features ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Type Ii ◽  
Hydrophobic Residues ◽  
P Glycoprotein ◽  
C Terminus ◽  
Signal Anchor ◽  
Hydrophilic Residues

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

scholarly journals The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology

1996 ◽  
Vol 319 (1) ◽  
pp. 131-136 ◽  
Author(s):  
Thomas FRIEDBERG ◽  
Romy HOLLER ◽  
Bettina LÖLLMANN ◽  
Michael ARAND ◽  
Franz OESCH
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Activity ◽  
Epoxide Hydrolase ◽  
Membrane Topology ◽  
Microsomal Epoxide Hydrolase ◽  
Type I ◽  
Type Ii ◽  
Membrane Orientation ◽  
N Terminus ◽  
Cytochrome P 450

Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial luminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.

The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

2000 ◽  
Vol 12 (7) ◽  
pp. 1179 ◽  
Author(s):  
Mohammed Benghezal ◽  
Geoffrey O. Wasteneys ◽  
David A. Jones
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Type I

scholarly journals Erv26p Directs Pro-Alkaline Phosphatase into Endoplasmic Reticulum–derived Coat Protein Complex II Transport Vesicles

2006 ◽  
Vol 17 (11) ◽  
pp. 4780-4789 ◽  
Author(s):  
Catherine A. Bue ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Protein ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Secretory Proteins ◽  
Complex Ii ◽  
Transport Vesicles ◽  
Copii Vesicles

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Δ mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER.

scholarly journals Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

2014 ◽  
Vol 206 (1) ◽  
pp. 79-95 ◽  
Author(s):  
Govind Kunduri ◽  
Changqing Yuan ◽  
Velayoudame Parthibane ◽  
Katherine M. Nyswaner ◽  
Ritu Kanwar ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Golgi Complex ◽  
G Protein Coupled Receptors ◽  
Phospholipase A1 ◽  
Copii Vesicles ◽  
G Protein Coupled

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

The fine structure of the developing macrogamete of Eimeria maxima

Parasitology ◽  
1979 ◽  
Vol 79 (2) ◽  
pp. 259-265 ◽  
Author(s):  
R. M. Pittilo ◽  
S. J. Ball
Keyword(s):  
Endoplasmic Reticulum ◽  
Fine Structure ◽  
Parasitophorous Vacuole ◽  
The Other ◽  
Granular Endoplasmic Reticulum ◽  
Type I ◽  
Type Ii ◽  
Eimeria Maxima ◽  
Lipid Inclusions

SUMMARYThe fine structure of the developing macrogamete of Eimeria maxima was studied from chicks killed at intervals from 138 to 147 h after inoculation. The macrogamete developed within a parasitophorous vacuole. Lying within this vacuole and extending for some distance around the periphery of the macrogamete were intravacuolar tubules, grouped in certain areas, and in some cases they were seen to make direct connexions with the cytoplasm of the parasite. During development, electron-pale vesicles were pinched off externally from the surface of the macrogamete. There appeared to be 2 forms of wall-forming bodies of the Type I during development, one form being less osmiophilic than the other. Other organelles present, such as wall-forming bodies of Type II, granular endoplasmic reticulum, mitochondria, canaliculi, lipid inclusions and intravacuolar folds, were similar in structure to those of other Eimeria species.

scholarly journals Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences

2013 ◽  
Vol 24 (19) ◽  
pp. 3123-3132 ◽  
Author(s):  
Takaaki Yabuki ◽  
Fumiko Morimoto ◽  
Yuichiro Kida ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Surface Plasmon Resonance Analysis ◽  
Binding Peptide ◽  
Resonance Analysis ◽  
N Terminus ◽  
Signal Anchor ◽  
Streptavidin Binding

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10−8 M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I–like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.

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