Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets

Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.     

Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER.     

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.     

A Hinge in the Distal End of the PACSIN 2 F-BAR Domain May Contribute to Membrane-Curvature Sensing

The protein kinase C and casein kinase 2 substrates in neurons (PACSINs) represent a subfamily of membrane-binding proteins characterized by an amino-terminal Bin-Amphiphysin-Rvs (F-BAR) domain. PACSINs link membrane trafficking with actin dynamics and regulate the localization of distinct cargo molecules. The F-BAR domain forms a dimer essential for lipid binding. We have obtained crystals of authentic murine PACSIN 2 that contain an ordered F-BAR domain, indicating that additional domains are flexibly connected to F-BAR. The structure shares similarity to other BAR domains and exhibits special features unique to PACSINs. These include the uneven distribution of charged residues on the concave molecular surface and a so-called wedge loop that is driven into the membrane upon binding of PACSIN. The murine PACSIN 2 F-BAR domain requires dimerization for sensing of curved membranes, and the present structure also provides a mechanism for higher-order oligomer formation. Importantly, comparison of murine with human and Drosophila PACSIN 2 F-BAR domains reveals stark differences in the orientation of distal helical segments leading to a wider crescent shape of murine PACSIN 2. We define hinge residues for these movements that may help PACSINs sense and concomitantly reinforce membrane curvature.     

Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. However, excessive accumulation of triacylglycerols in adipose tissue leads to obesity and, in nonadipose tissues, is associated with tissue dysfunction. Hence, it is of great importance to have a better understanding of the molecular mechanisms of triacylglycerol synthesis. The final step in triacylglycerol synthesis is catalyzed by the acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although recent studies have shed light on metabolic functions of these enzymes, little is known about the molecular aspects of their structures or functions. Here we report the topology for murine DGAT2 and the identification of key amino acids that likely contribute to enzymatic function. Our data indicate that DGAT2 is an integral membrane protein with both the N and C termini oriented toward the cytosol. A long hydrophobic region spanning amino acids 66-115 likely comprises two transmembrane domains or, alternatively, a single domain that is embedded in the membrane bilayer. The bulk of the protein lies distal to the transmembrane domains. This region shares the highest degree of homology with other enzymes of the DGAT2 family and contains a sequence HPHG that is conserved in all family members. Mutagenesis of this sequence in DGAT2 demonstrated that it is required for full enzymatic function. Additionally, a neutral lipid-binding domain that is located in the putative first transmembrane domain was also required for full enzymatic function. Our findings provide the first insights into the topography and molecular aspects of DGAT2 and related enzymes.     

Two Cooperating Helices Constitute the Lipid-binding Domain of the Bacterial SRP Receptor

Protein targeting by the bacterial signal recognition particle requires the specific interaction of the signal recognition particle (SRP)-ribosome-nascent chain complex with FtsY, the bacterial SRP receptor. Although FtsY in Escherichia coli lacks a transmembrane domain, the membrane-bound FtsY displays many features of an integral membrane protein. Our data reveal that it is the cooperative action of two lipid-binding helices that allows this unusually strong membrane contact. Helix I comprises the first 14 amino acids of FtsY and the second is located at the interface between the A- and the N-domain of FtsY. We show by site-directed cross-linking and binding assays that both helices bind to negatively charged phospholipids, with a preference for phosphatidyl glycerol. Despite the strong lipid binding, helix I does not seem to be completely inserted into the lipid phase, but appears to be oriented parallel with the membrane surface. The two helices together with the connecting linker constitute an independently folded domain, which maintains its lipid binding even in the absence of the conserved NG-core of FtsY. In summary, our data reveal that the two consecutive lipid-binding helices of FtsY can provide a membrane contact that does not differ significantly in stability from that provided by a transmembrane domain. This explains why the bacterial SRP receptor does not require an integral β-subunit for membrane binding.     

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ∼50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation.     

Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix

The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.     

Intrinsically Disordered Linker and Plasma Membrane‐Binding Motif Sort Ist2 and Ssy1 to Junctions

Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)‐resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM‐binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites.      

The presenilin C-terminus is required for ER-retention, nicastrin-binding and gamma-secretase activity

gamma-Secretase is an intramembrane cleaving protease involved in Alzheimer's disease. gamma-Secretase occurs as a high molecular weight complex composed of presenilin (PS1/2), nicastrin (NCT), anterior pharynx-defective phenotype 1 and PS enhancer 2. Little is known about the cellular mechanisms of gamma-secretase assembly. Here we demonstrate that the cytoplasmic tail of PS1 fulfills several functions required for complex formation, retention of unincorporated PS1 and gamma-secretase activity. The very C-terminus interacts with the transmembrane domain of NCT and may penetrate into the membrane. Deletion of the last amino acid is sufficient to completely block gamma-secretase assembly and release of PS1 from the endoplasmic reticulum (ER). This suggests that unincorporated PS1 is actively retained within the ER. We identified a hydrophobic stretch of amino acids within the cytoplasmic tail of PS1 distinct from the NCT-binding site, which is required to retain unincorporated PS1 within the ER. Deletion of the retention signal results in the release of PS1 from the ER and the assembly of a nonfunctional gamma-secretase complex, suggesting that at least a part of the retention motif may also be required for the function of PS1.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

The Endoplasmic Reticulum Enzyme DGAT2 Is Found in Mitochondria-associated Membranes and Has a Mitochondrial Targeting Signal That Promotes Its Association with Mitochondria

The synthesis and storage of neutral lipids in lipid droplets is a fundamental property of eukaryotic cells, but the spatial organization of this process is poorly understood. Here we examined the intracellular localization of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), an enzyme that catalyzes the final step of triacylglycerol (TG) synthesis in eukaryotes. We found that DGAT2 expressed in cultured cells localizes to the endoplasmic reticulum (ER) under basal conditions. After providing oleate to drive TG synthesis, DGAT2 also localized to near the surface of lipid droplets, where it co-localized with mitochondria. Biochemical fractionation revealed that DGAT2 is present in mitochondria-associated membranes, specialized domains of the ER that are highly enriched in lipid synthetic enzymes and interact tightly with mitochondria. The interaction of DGAT2 with mitochondria depended on 67 N-terminal amino acids of DGAT2, which are not conserved in family members that have different catalytic functions. This targeting signal was sufficient to localize a red fluorescent protein to mitochondria. A highly conserved, positively charged, putative mitochondrial targeting signal was identified in murine DGAT2 between amino acids 61 and 66. Thus, DGAT2, an ER-resident transmembrane domain-containing enzyme, is also found in mitochondria-associated membranes, where its N terminus may promote its association with mitochondria.Most eukaryotic cells can synthesize neutral lipids, such as triacylglycerols (TGs)² and sterol esters, and store them in cytosolic lipid droplets. Yet, a molecular understanding of this process and how it is spatially organized is lacking. For example, lipid substrates for TG synthesis (fatty acids and glycerolipid precursors) are found in the cytoplasm and membranes, energy for activating fatty acids (by converting to fatty acyl-CoA) comes from mitochondria, and the enzymes that catalyze TG formation are primarily found in the mitochondria and endoplasmic reticulum (ER). How the cell orchestrates this complex anabolic process to maximize lipid synthesis and storage during times of substrate excess is poorly understood.In most cells, TG synthesis occurs via the glycerol 3-phosphate (Kennedy) pathway and involves multiple enzymatic reactions in different subcellular compartments (1). The fatty acids for TG synthesis must first be “activated” by acyl-CoA synthases, a family of enzymes that localize to membranes of different compartments, including the ER, mitochondria, and plasma membrane (2), and utilize ATP to ligate CoA to the fatty acyl chain. Next, these fatty acids enter the Kennedy pathway of glycerolipid synthesis, in which the first two reactions occur in both the ER and mitochondria. In the first reaction, glycerol 3-phosphate and a fatty acyl-CoA are combined to yield lysophosphatidic acid through the actions of glycerol-3-phosphate acyltransferase enzymes (1, 3). In the second reaction, 1-acylglycerol-3-phosphate O-acyltransferase enzymes catalyze the esterification of lysophosphatidic acid with fatty acyl-CoA to form phosphatidic acid (1, 4). Next, phosphatidic acid is dephosphorylated at membrane surfaces by phosphatidate phosphatase to yield diacylglycerol (1, 5, 6). All these steps are highly organized spatially, which is likely to be important for the efficiency of the pathway.The final reaction of TG synthesis is catalyzed by acyl-CoA: diacylglycerol acyltransferase (DGAT) enzymes (7-9). The two mammalian DGATs, DGAT1 and DGAT2 (10, 11), which are encoded by genes of different families, have distinct roles in TG synthesis (12). DGAT2 is the major TG biosynthetic enzyme in eukaryotes. Dgat2-deficient mice die shortly after birth and are almost completely devoid of TG (13), indicating an essential requirement for DGAT2. Catalysis of TG synthesis is conserved in the DGAT2 gene family, with functional orthologs in many species, including Dga1p in Saccharomyces cerevisiae, which contributes to a major portion of TG synthesis (14-16).Little is known about the intracellular localization of DGAT enzymes. DGAT activity is present in microsomes (7, 17, 18), but in vitro assays do not distinguish between DGAT1 and DGAT2. A DGAT2-green fluorescent fusion protein expressed in HeLa cells localized to the ER (19), and Dga1p activity in S. cerevisiae localizes to the ER and lipid droplets (16). DGAT1 and DGAT2 expressed in COS-7 cells localized primarily to the ER (20). A recent study of the subcellular localizations of tung tree DGAT1 and DGAT2 in tobacco BY-2 cells revealed that the enzymes are located in distinct, non-overlapping regions of the ER (21). Most recently, DGAT2 was reported to co-localize with lipid droplets in cultured adipocytes (22). As a step toward a better understanding of the cellular organization of processes that contribute to TG synthesis and storage, we determined the subcellular localization of murine DGAT2 in mammalian cells.     

Membrane topology and identification of key residues of EaDAcT,a plant MBOAT with unusual substrate specificity

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to the sn‐3 position of diacylglycerol to form 3‐acetyl‐1,2‐diacyl‐sn‐glycerol (acetyl‐TAG). EaDAcT belongs to a small, plant‐specific subfamily of the membrane bound O‐acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl‐TAG‐producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.     

Hydrophobic-domain-dependent protein-protein interactions mediate the localization of GPAT enzymes to ER subdomains

The endoplasmic reticulum (ER) is a dynamic organelle that consists of numerous regions or 'subdomains' that have discrete morphological features and functional properties. Although it is generally accepted that these subdomains differ in their protein and perhaps lipid compositions, a clear understanding of how they are assembled and maintained has not been well established. We previously demonstrated that two diacylglycerol acyltransferase enzymes (DGAT1 and DGAT2) from tung tree (Vernicia fordii) were located in different subdomains of ER, but the mechanisms responsible for protein targeting to these subdomains were not elucidated. Here we extend these studies by describing two glycerol-3-phosphate acyltransferase-like (GPAT) enzymes from tung tree, GPAT8 and GPAT9, that both colocalize with DGAT2 in the same ER subdomains. Measurement of protein-protein interactions using the split-ubiquitin assay revealed that GPAT8 interacts with itself, GPAT9 and DGAT2, but not with DGAT1. Furthermore, mutational analysis of GPAT8 revealed that the protein's first predicted hydrophobic region, which contains an amphipathic helix-like motif, is required for interaction with DGAT2 and for DGAT2-dependent colocalization in ER subdomains. Taken together, these results suggest that the regulation and organization of ER subdomains is mediated at least in part by higher-ordered, hydrophobic-domain-dependent homo- and hetero-oligomeric protein-protein interactions.     

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.     

Characterization of the C-terminal ER membrane anchor of PTP1B

The tyrosine phosphatase PTP1B is an important regulator of cell function. In living cells PTP1B activity is restricted to the vicinity of the endoplasmic reticulum (ER) by post-translational C-terminal attachment of PTP1B to the ER membrane network. In our study we investigated the membrane anchor of PTP1B by use of EGFP fusion proteins. We demonstrate that the membrane anchor of PTP1B cannot be narrowed down to a unique amino acid sequence with a defined start and stop point but rather is moveable within several amino acids. Removal of up to seven amino acids from the C-terminus, as well as exchange of single amino acids in the putative transmembrane sequence did not influence subcellular localization of PTP1B. With the method of bimolecular fluorescence complementation we could demonstrate dimerization of PTP1B in vivo. Homodimerization was, in contrast to other tail-anchored proteins, not dependent on the membrane anchor. Our data demonstrate that the C-terminal membrane anchor of PTP1B is formed by a combination of a single stretch transmembrane domain (TMD) followed by a tail. TMD and tail length are variable and there are no sequence-specific features. Our data for PTP1B are consistent with a concept that explains the ER membrane anchor of tail-anchored proteins as a physicochemical structure.     

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

p38: A novel protein that associates with the vesicular stomatitis virus glycoprotein

The vesicular stomatitis virus glycoprotein (VSV G) is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. The cytoplasmic domain of VSV G contains information for several intracellular sorting steps including efficient export from the ER, basolateral delivery, and endocytosis. In order to identify proteins that potentially interact with the polypeptide sorting motifs in the VSV G tail, the carboxy-terminal 27 amino acids of VSV G were used as bait in a yeast two-hybrid system. The protein identified most frequently in the screen is a novel protein of 38 kDa, p38. In the present work, the initial molecular and biochemical characterization of p38 is described. Preliminary evidence suggests that p38 may interact transiently with endoplasmic reticulum (ER) membranes, and thus may affect VSV G and other cargo movement at the step of ER to Golgi traffic.