apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB

ABSTRACTMouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the ER membrane into a functional N-terminal signal peptide (SP) and the C-terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, yet was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug Brefeldin A (BFA), which interferes with the ER to Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels. A Rem mutant lacking glycosylation sites was cleaved into SP and Rem-CT, but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not exit the ER or reach a degradative compartment. BFA reduction of Rem-CT levels was not rescued by proteasome or lysosomal inhibitors. Rem-CT has simple glycans, which are necessary for Rem-CT stability and trafficking, but indicate that Rem-CT does not traffic through the Golgi. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, were co-localized with Rab5+ endosomes. Expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (T31N) mimicked the effects of BFA by reducing Rem-CT levels, suggesting that Arf1 prevents Rem-CT localization to a degradative compartment. A DN form of the AAA ATPase, p97/VCP, rescued Rem-CT in the presence of BFA or DN Arf1. Thus, Rem-CT uses an unconventional trafficking scheme, perhaps to thwart innate immunity to MMTV infection.IMPORTANCEMouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP eludes ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used multiple methods to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation for trafficking to endosomes. Blocking of retrograde trafficking through Arf1 reduced Rem-CT levels, but was not restored by lysosomal or proteasomal inhibitors. The unique trafficking of Rem-CT suggests a novel intracellular trafficking pathway, potentially impacting host anti-viral immunity.

Download Full-text

A disease-associated frameshift mutation in caveolin-1 disrupts caveolae formation and function through introduction of a de novo ER retention signal

Molecular Biology of the Cell ◽

10.1091/mbc.e17-06-0421 ◽

2017 ◽

Vol 28 (22) ◽

pp. 3095-3111 ◽

Cited By ~ 11

Author(s):

Courtney A. Copeland ◽

Bing Han ◽

Ajit Tiwari ◽

Eric D. Austin ◽

James E. Loyd ◽

...

Keyword(s):

Frameshift Mutation ◽

De Novo ◽

Dominant Negative ◽

Caveolin 1 ◽

Protein Levels ◽

C Terminus ◽

Er Retention ◽

And Function ◽

Er Retention Signal ◽

Retention Signal

Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1–/– MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1–/– MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.

Download Full-text

An NMDA Receptor ER Retention Signal Regulated by Phosphorylation and Alternative Splicing

Journal of Neuroscience ◽

10.1523/jneurosci.21-09-03063.2001 ◽

2001 ◽

Vol 21 (9) ◽

pp. 3063-3072 ◽

Cited By ~ 274

Author(s):

Derek B. Scott ◽

Thomas A. Blanpied ◽

Geoffrey T. Swanson ◽

Chi Zhang ◽

Michael D. Ehlers

Keyword(s):

Alternative Splicing ◽

Nmda Receptor ◽

Er Retention ◽

Er Retention Signal ◽

Retention Signal

Download Full-text

Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe.

The EMBO Journal ◽

10.1002/j.1460-2075.1992.tb05203.x ◽

1992 ◽

Vol 11 (4) ◽

pp. 1583-1591 ◽

Cited By ~ 45

Author(s):

A.L. Pidoux ◽

J. Armstrong

Keyword(s):

Schizosaccharomyces Pombe ◽

Er Retention ◽

Er Retention Signal ◽

Retention Signal

Download Full-text

Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis

Plant Molecular Biology ◽

10.1007/s11103-012-9902-5 ◽

2012 ◽

Vol 79 (1-2) ◽

pp. 157-169 ◽

Cited By ~ 19

Author(s):

Xu He ◽

Thomas Haselhorst ◽

Mark von Itzstein ◽

Daniel Kolarich ◽

Nicolle H. Packer ◽

...

Keyword(s):

Er Retention ◽

Er Retention Signal ◽

Retention Signal

Download Full-text

An orderly inactivation of intracellular retention signals controls surface expression of the T cell antigen receptor

Journal of Experimental Medicine ◽

10.1084/jem.20041133 ◽

2005 ◽

Vol 201 (4) ◽

pp. 555-566 ◽

Cited By ~ 27

Author(s):

Pilar Delgado ◽

Balbino Alarcón

Keyword(s):

Plasma Membrane ◽

T Cell ◽

Cell Surface ◽

Cell Receptor ◽

Surface Expression ◽

Membrane Receptors ◽

Membrane Expression ◽

Er Retention ◽

Er Retention Signal ◽

Retention Signal

Exit from the endoplasmic reticulum (ER) is an important checkpoint for proper assembly of multimeric plasma membrane receptors. The six subunits of the T cell receptor (TCR; TCRα, TCRβ, CD3γ, CD3δ, CD3ε, and CD3ζ) are each endowed with ER retention/retrieval signals, and regulation of its targeting to the plasma membrane is therefore especially intriguing. We have studied the importance of the distinct ER retention signals at different stages of TCR intracellular assembly. To this end, we have characterized first the presence of ER retention signals in CD3γ. Despite the presence of multiple ER retention signals in CD3γ, εγ dimers reach the cell surface when the single CD3ε ER retention signal is deleted. Furthermore, inclusion of this CD3ε mutant promoted plasma membrane expression of incomplete αβγε and αβδε complexes without CD3ζ. It therefore appears that the CD3ε ER retention signal is dominant and that it is only overridden upon the incorporation of CD3ζ. We propose that the stepwise assembly of the TCR complex guarantees that all assembly intermediates have at least one functional ER retention signal and that only a full signaling-competent TCR complex is expressed on the cell surface.

Download Full-text

Characterization of membrane topology and retention signal of pestiviral glycoprotein E1

Journal of Virology ◽

10.1128/jvi.00521-21 ◽

2021 ◽

Author(s):

Yu Mu ◽

Christina Radtke ◽

Birke Andrea Tews ◽

Gregor Meyers

Keyword(s):

Middle Part ◽

Membrane Topology ◽

Signal Peptidase ◽

Type I ◽

Diarrhea Virus ◽

Er Retention ◽

Polar Residues ◽

And Function ◽

Er Retention Signal ◽

Retention Signal

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1 and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface, but localizes predominantly in the ER. Using engineered chimeric TM domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labelling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C-terminus whereas it adopts a hairpin-like structure with the C-terminus located in the ER lumen when the pre-cleavage situation is mimicked by blocking the cleavage site between E1 and E2. Importance The shortage of specific antibodies against E1 making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the TM domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy-terminus located on the luminal side of the ER in the pre-cleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

Download Full-text

A novel ER-derived compartment, the ER body, selectively accumulates a β-glucosidase with an ER-retention signal inArabidopsis

The Plant Journal ◽

10.1046/j.1365-313x.2003.01636.x ◽

2003 ◽

Vol 33 (3) ◽

pp. 493-502 ◽

Cited By ~ 110

Author(s):

Ryo Matsushima ◽

Maki Kondo ◽

Mikio Nishimura ◽

Ikuko Hara-Nishimura

Keyword(s):

Er Retention ◽

Er Retention Signal ◽

Retention Signal

Download Full-text

Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen.

The Journal of Cell Biology ◽

10.1083/jcb.133.2.469 ◽

1996 ◽

Vol 133 (2) ◽

pp. 469-483 ◽

Cited By ~ 145

Author(s):

M Satoh ◽

K Hirayoshi ◽

S Yokota ◽

N Hosokawa ◽

K Nagata

Keyword(s):

Triple Helix ◽

Secretory Pathway ◽

Stress Protein ◽

Biological Significance ◽

Confocal Laser ◽

Er Retention ◽

Polypeptide Chains ◽

Er Retention Signal ◽

Retention Signal ◽

Kdel Sequence

Heat shock protein 47 (HSP47), a collagen-specific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER. We previously demonstrated that HSP47 transiently associated with newly synthesized procollagen in the ER (Nakai, A., M. Satoh, K. Hirayoshi, and K. Nagata. 1992. J. Cell Biol. 117:903-914). In the present work, we examined the location where HSP47 binds to and dissociates from newly synthesized procollagen within the cells, and whether HSP47 associates with nascent single procollagen polypeptide chains and/or with mature triple-helix procollagen. This was accomplished by biochemical coprecipitation with anti-HSP47 and anticollagen antibodies, combined with pulse-label and chase experiments in the presence or absence of various inhibitors for protein secretion, as well as by confocal laser microscopic observation of the cells double stained with both antibodies. We further examined whether the RDEL (Arg-Asp-Glu-Leu) sequence at the COOH terminus of HSP47 can act as an ER-retention signal, as the KDEL sequence does. When the secretion of procollagen was inhibited by the presence of alpha, alpha'-dipyridyl, an iron chelator that inhibits procollagen triple-helix formation, or by the presence of brefeldin A. which inhibits protein transport between the ER and the Golgi apparatus, procollagen was found to be bound to HSP47 during the chase period in the intermediate compartment. In contrast, the dissociation of procollagen chains from HSP47 was not inhibited when procollagen secretion was inhibited by monensin or bafilomycin A1, both of which are known to be inhibitors of post-cis-Golgi transport. These findings suggest that HSP47 and procollagen dissociated between the post-ER and the cis-Golgi compartments. HSP47 was shown to bind to nascent, single-polypeptide chains of newly synthesized procollagen, as well as to the mature triple-helix form of procollagen. HSP47 with the RDEL sequence deleted was secreted out of the cells, which suggests that the RDEL sequence actually acts as an ER-retention signal, as the KDEL sequence does. This secreted HSP47 did not acquire endoglycosidase H resistance. The biological significance of the interaction between HSP47 and procollagen in the central secretory pathway, as well as possible mechanisms for this pathway, will be discussed.

Download Full-text