Lysine can be replaced by histidine but not by arginine as the ER retrieval motif for type I membrane proteins

The OST48 subunit of the oligosaccharyltransferase complex is a type I membrane protein containing three lysines in its cytosolic domain. The two lysines in positions 3 and 5 from the C-terminus are able to direct protein localisation within the endoplasmic reticulum (ER) by COPI-mediated retrieval. Substitution of these lysines by arginine resulted in cell-surface expression of OST48, whereas ER residency was maintained when either Lys-5 or Lys-3 but not both was replaced with arginine. Localisation of OST48 was not affected by substitution of the two lysines by histidine, indicating that a His-Xaa-His sequence, in contrast to Arg-Xaa-Arg, contains ER-specific targeting information. These differences show that simple charge interactions are not sufficient for ER retention and that other structural factors also play a role. The His-Xaa-His sequence could represent a new and independent signal for directing ER localisation differing from both the arginine motif in type II proteins and the lysine motif in type I proteins. Our data do not exclude, however, that the histidine sequence simply mimics the lysine motif as a sorting signal, being recognised by and interacting with the same receptor subunit(s) in COP-I-coated vesicles. Conclusions arising from this assumption involving the conformation of lysine at the putative COP-I binding site and the failure of Arg-Xaa-Arg to mediate ER localisation for type I proteins are discussed.     

An N-terminal diacidic motif is required for the trafficking of maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the plasma membrane

Maize plasma membrane aquaporins (ZmPIPs, where PIP is the plasma membrane intrinsic protein) fall into two groups, ZmPIP1s and ZmPIP2s, which, when expressed alone in mesophyll protoplasts, are found in different subcellular locations. Whereas ZmPIP1s are retained in the endoplasmic reticulum (ER), ZmPIP2s are found in the plasma membrane (PM). We previously showed that, when co-expressed with ZmPIP2s, ZmPIP1s are relocalized to the PM, and that this relocalization results from the formation of hetero-oligomers between ZmPIP1s and ZmPIP2s. To determine the domains responsible for the ER retention and PM localization, respectively, of ZmPIP1s and ZmPIP2s, truncated and mutated ZmPIPs were generated, together with chimeric proteins created by swapping the N- or C-terminal regions of ZmPIP2s and ZmPIP1s. These mutated proteins were fused to the mYFP and/or mCFP, and the fusion proteins were expressed in maize mesophyll protoplasts, and were then localized by microscopy. This allowed us to identify a diacidic motif, DIE (Asp-Ile-Glu), at position 4–6 of the N-terminus of ZmPIP2;5, that is essential for ER export. This motif was conserved and functional in ZmPIP2;4, but was absent in ZmPIP2;1. In addition, we showed that the N-terminus of ZmPIP2;5 was not sufficient to cause the export of ZmPIP1;2 from the ER. A study of ZmPIP1;2 mutants suggested that the N- and C-termini of this protein are probably not involved in ER retention. Together, these results show that the trafficking of maize PM aquaporins is differentially regulated depending on the isoform, and involves a specific signal and mechanism.     

The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions and undergoes constant remodeling through formation and loss of the three-way junctions. Transmembrane and coiled-coil domain family 3 (TMCC3), an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N terminus and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wildtype TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.     

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1(ts)) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.     

The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake

Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leaflet of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced by RNA interference of ACSL3. In conclusion, the structural organization of ACSL3 allows the fast and efficient movement from the ER to emerging LDs. ACSL3 not only esterifies fatty acids with CoA but is also involved in the cellular uptake of fatty acids, presumably indirectly by metabolic trapping. The unique localization of the acyl-CoA synthetase ACSL3 on LDs suggests a function in the local synthesis of lipids.     

Endoplasmic reticulum protein HSP47 binds specifically to the N-Terminal globular domain of the amino-propeptide of the procollagen I α1(I)-chain

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with³⁵[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro α1 (l)-N-propeptide at 25 μg/ml compeletely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86–90 of the pro α1 (l)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro α1 (l)-N-propeptide domain were confirmed by examination of polysome-associated pro α chains. The nascent pro α1(l)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro α1 (l)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23–108 (NP1), 23–151 (NP2), and 23–178 (NP3) within the pro α1 (l)-N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro α1(l)- and pro α2(l)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro α2(l)-chains and hsp47, whereas there was strong interaction between pro α1 (l)-chains and nascent hsp47. SP1.D8 could not precipitate pro α1 (l)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a “chaperone” role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the proα1 (l) chain, and the pro α1 (l) amino-propeptide region in particular. © 1995 Wiley-Liss, Inc.     

The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding

The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes. This reaction requires an efficient membrane binding, and the N-terminal beta-barrel domain of the enzyme has been implicated in this process. To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wild-type 15-lipoxygenase, its beta-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics. We found that: (i) the beta-barrel deletion mutant was catalytically active and its enzymatic properties (K(M), V(max), pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wild-type lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the beta-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding. Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme. These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal beta-barrel and the C-terminal catalytic domain.     

Tryptophan‐rich basic protein (WRB) mediates insertion of the tail‐anchored protein otoferlin and is required for hair cell exocytosis and hearing

The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail‐anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan‐rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb‐deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca²⁺ channels, and membrane‐proximal vesicles, but contained fewer ribbon‐associated vesicles. Patch‐clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use‐dependent reduction in sound‐evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.     

A Functional GTPase Domain,but not its Transmembrane Domain,is Required for Function of the SRP Receptor β-subunit

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRα and SRβ, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRβ. This notion is supported (a) by Srp102p''s sequence similarity to SRβ; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRα homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.     

Urban planning of the endoplasmic reticulum (ER): How diverse mechanisms segregate the many functions of the ER

The endoplasmic reticulum (ER) is the biggest organelle in most cell types, but its characterization as an organelle with a continuous membrane belies the fact that the ER is actually an assembly of several, distinct membrane domains that execute diverse functions. Almost 20 years ago, an essay by Sitia and Meldolesi first listed what was known at the time about domain formation within the ER. In the time that has passed since, additional ER domains have been discovered and characterized. These include the mitochondria-associated membrane (MAM), the ER quality control compartment (ERQC), where ER-associated degradation (ERAD) occurs, and the plasma membrane-associated membrane (PAM). Insight has been gained into the separation of nuclear envelope proteins from the remainder of the ER. Research has also shown that the biogenesis of peroxisomes and lipid droplets occurs on specialized membranes of the ER. Several studies have shown the existence of specific marker proteins found on all these domains and how they are targeted there. Moreover, a first set of cytosolic ER-associated sorting proteins, including phosphofurin acidic cluster sorting protein 2 (PACS-2) and Rab32 have been identified. Intra-ER targeting mechanisms appear to be superimposed onto ER retention mechanisms and rely on transmembrane and cytosolic sequences. The crucial roles of ER domain formation for cell physiology are highlighted with the specific targeting of the tumor metastasis regulator gp78 to ERAD-mediating membranes or of the promyelocytic leukemia protein to the MAM.     

Role of the Lectin VIP36 in Post‐ER Quality Control of Human α1‐Antitrypsin

The leguminous‐type (L‐type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high‐mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar‐binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein α1‐antitrypsin (α1‐AT) as a cargo of VIP36. The VIP36/α1‐AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high‐mannose form of α1‐AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of α1‐AT were inactivated by mutagenesis. Silencing VIP36 accelerated α1‐AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and α1‐AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post‐ER quality control of α1‐AT.     

The N-terminal basic domain of the HIV-1 matrix protein does not contain a conventional nuclear localization sequence but is required for DNA binding and protein self-association

The HIV p17 or matrix (MA) protein has long been implicated in the process of nuclear import of the HIV genome and thus the ability of the virus to infect nondividing cells such as macrophages. While it has been demonstrated that MA is not absolutely required for this process, debate continues to surround the subcellular targeting properties of MA and its potential contribution to nuclear import of the HIV cDNA. Through the use of in vitro techniques we have determined that, despite the ability of MA to interact with importins, the full-length protein fails to enter the nucleus of cells. While MA does contain a region of basic amino acids within its N-terminus which can confer nuclear accumulation of a fusion protein, we show that this is due to nuclear retention mediated by DNA binding and does not represent facilitated import. Importantly, we show that the 26KK residues of MA, previously thought to be part of a nuclear localization sequence, are absolutely required for a number of MA's functions including its ability to bind DNA and RNA and its propensity to form high-order multimers/protein aggregates. The results presented here indicate that the N-terminal basic domain of MA does not appear likely to play a role in HIV cDNA nuclear import; rather this region appears to be a crucial structural and functional motif whose integrity is required for a number of other roles performed by MA during viral infection.     

ATP is required for in vitro assembly of MHC class I antigens but not for transfer of peptides across the ER membrane

We have translated the HLA-B27 heavy chain in vitro and studied its assembly with beta 2-microglobulin and peptide in microsomes from human cells. The assembly process requires ATP. However, the translocation of peptide across the endoplasmic reticulum (ER) membrane does not require ATP, and binding of biotinylated peptide to BiP, an ER luminal protein, occurs after ATP depletion. Proteinase K treatment of the microsomes does not block peptide translocation. Thus, ATP is required in the lumen of the ER for efficient assembly to occur. Microsomes prepared from Raji and T1 cells show similar levels of assembly, whereas assembly in T2 microsomes is 10-fold lower. This difference remains after peptide stimulation of assembly. The inefficient assembly in T2 microsomes is not due to impaired peptide translocation across the ER membrane, as no difference was found compared with microsomes from T1 cells. Instead, the defect seems to reside in the lumen of the ER.     

Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

Role of H(abc) domain in membrane trafficking and targeting of syntaxin 1A

Membrane syntaxin plays essential roles in exocytosis in eukaryotic cells. The conservative H(abc) domain in plasma membrane syntaxins implies important roles for syntaxin targeting and function. Our previous study showed H(abc) domain was necessary for the trafficking and cluster distribution of syntaxin 1A on the plasma membrane. Here we identified which of the three domains (H(a), H(b) and H(c)) was essential for Stx1A trafficking and clustering. We found that, in INS-1 cells, the mutant truncated with either H(a), H(b) or H(c) domain could be sorted to the cell surface by a different mechanism compared to that of whole H(abc) truncated mutant. In contrast to wild type Stx1A, none of the mutants showed cluster distribution at the functional sites, suggesting that the physiological localization of Stx1A relies on intact H(abc) domain. Furthermore Munc18-1 is found not to be essential for Stx1A cluster distribution, despite important role in stabilizing membrane delivery of Stx1A.     

The octarepeat region of prion protein, but not the TM1 domain, is important for the antioxidant effect of prion protein

The cellular prion protein (PrP(c)) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP(c) (residues 110-135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N(2)A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP(c) (wt-PrP) or a deletion mutant affecting TM1 (Delta8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Deltaocta-PrP). Overexpression of wt-PrP, Delta8TM1-PrP, or Deltaocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Deltaocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP(c) against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.