Formation of Crystalloid Endoplasmic Reticulum Induced by Expression of Synaptotagmin Lacking the Conserved WHXL Motif in the C Terminus

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

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Mechanism for antigenic peptide selection by endoplasmic reticulum aminopeptidase 1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912070116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 26709-26716 ◽

Cited By ~ 7

Author(s):

Petros Giastas ◽

Anastasia Mpakali ◽

Athanasios Papakyriakou ◽

Aggelos Lelis ◽

Paraskevi Kokkala ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Structural Motif ◽

Internal Cavity ◽

Sequence Specificity ◽

Substrate Selection ◽

Class I Mhc ◽

Mhc I ◽

Intracellular Enzyme ◽

C Terminus ◽

Endoplasmic Reticulum Aminopeptidase 1

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.

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Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.9.9.2681 ◽

1998 ◽

Vol 9 (9) ◽

pp. 2681-2697 ◽

Cited By ~ 29

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.

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Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805757115 ◽

2018 ◽

Vol 115 (31) ◽

pp. E7331-E7340 ◽

Cited By ~ 46

Author(s):

Ben Johnson ◽

Ashley N. Leek ◽

Laura Solé ◽

Emily E. Maverick ◽

Tim P. Levine ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Neuronal Cultures ◽

Physical Relationship ◽

Membrane Contact Sites ◽

Contact Sites ◽

Hek Cells ◽

C Terminus ◽

Membrane Contact

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

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TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2231 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2231-2247 ◽

Cited By ~ 80

Author(s):

Julia D. Romano ◽

Walter K. Schmidt ◽

Susan Michaelis

Keyword(s):

Endoplasmic Reticulum ◽

Subcellular Fractionation ◽

Endoplasmic Reticulum Membrane ◽

Carboxyl Methylation ◽

C Terminus ◽

Eukaryotic Proteins ◽

Internal Site ◽

Membrane Spanning ◽

Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

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Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0463 ◽

2011 ◽

Vol 22 (24) ◽

pp. 4726-4739 ◽

Cited By ~ 37

Author(s):

Noa Furth ◽

Or Gertman ◽

Ayala Shiber ◽

Omri S. Alfassy ◽

Itamar Cohen ◽

...

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Protein Folding ◽

Nuclear Envelope ◽

Reverse Genetics ◽

Point Mutations ◽

Hydrophobic Surface ◽

Structural Features ◽

Er Quality Control ◽

C Terminus

Proper functioning of the protein-folding quality control network depends on the network's ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell homeostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control–associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone–dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.

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Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST

The Journal of Cell Biology ◽

10.1083/jcb.201301031 ◽

2013 ◽

Vol 201 (1) ◽

pp. 81-95 ◽

Cited By ~ 54

Author(s):

Shiteshu Shrimal ◽

Steven F. Trueman ◽

Reid Gilmore

Keyword(s):

Endoplasmic Reticulum ◽

Positive Selection ◽

Protein C ◽

Negative Selection ◽

Bioinformatics Analysis ◽

Chain Termination ◽

Catalytic Subunits ◽

C Terminus ◽

Glycosylation Sites ◽

Selection Of

Metazoan organisms assemble two isoforms of the oligosaccharyltransferase (OST) that have different catalytic subunits (STT3A or STT3B) and partially nonoverlapping roles in asparagine-linked glycosylation. The STT3A isoform of the OST is primarily responsible for co-translational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The C-terminal 65–75 residues of a glycoprotein will not contact the translocation channel–associated STT3A isoform of the OST complex before chain termination. Biosynthetic pulse labeling of five human glycoproteins showed that extreme C-terminal glycosylation sites were modified by an STT3B-dependent posttranslocational mechanism. The boundary for STT3B-dependent glycosylation of C-terminal sites was determined to fall between 50 and 55 residues from the C terminus of a protein. C-terminal NXT sites were glycosylated more rapidly and efficiently than C-terminal NXS sites. Bioinformatics analysis of glycopeptide databases from metazoan organisms revealed a lower density of C-terminal acceptor sites in glycoproteins because of reduced positive selection of NXT sites and negative selection of NXS sites.

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TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER–Golgi intermediate compartments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1709120114 ◽

2017 ◽

Vol 114 (37) ◽

pp. E7707-E7716 ◽

Cited By ~ 29

Author(s):

Michael G. Hanna ◽

Samuel Block ◽

E. B. Frankel ◽

Feng Hou ◽

Adam Johnson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Coat Protein ◽

Protein Trafficking ◽

Secretory Protein ◽

Dependent Manner ◽

Outer Coat ◽

Concentration Dependent Manner ◽

C Terminus ◽

Intermediate Compartment ◽

Fused Gene

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER–Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

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The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

Biochemical Journal ◽

10.1042/bj20060990 ◽

2007 ◽

Vol 401 (3) ◽

pp. 701-709 ◽

Cited By ~ 13

Author(s):

Matthew P. A. Henderson ◽

Yeen Ting Hwang ◽

John M. Dyer ◽

Robert T. Mullen ◽

David W. Andrews

Keyword(s):

Endoplasmic Reticulum ◽

Subcellular Localization ◽

Lipid Bilayers ◽

Fusion Proteins ◽

Molecular Mechanisms ◽

Cytochrome B5 ◽

Terminal Sequence ◽

C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

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Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.10.2.329 ◽

1999 ◽

Vol 10 (2) ◽

pp. 329-344 ◽

Cited By ~ 44

Author(s):

Martin Dünnwald ◽

Alexander Varshavsky ◽

Nils Johnsson

Keyword(s):

Endoplasmic Reticulum ◽

Protein Interactions ◽

Polypeptide Chain ◽

Protein Translocation ◽

Signal Sequence ◽

Living Cells ◽

C Terminus ◽

Identical Test ◽

Split Ubiquitin

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum ofSaccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

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