Alpha Beta Gamma Delta Epsilon Research Articles

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI‐1

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

Regulation of selective protein degradation in the endoplasmic reticulum by redox potential.

Recent studies show that the endoplasmic reticulum (ER) contains proteases, but it is not understood how these enzymes are regulated. In this report we study the selective ER degradation of the subunits (alpha beta gamma delta epsilon zeta) of the T-cell antigen receptor (TCR). When analyzed in vivo, unassembled subunits of the TCR fail to reach the Golgi apparatus and show a differential sensitivity to degradation after synthesis. The alpha, beta, and delta subunits are degraded rapidly, while gamma, epsilon, and zeta are stable. To study the regulation of proteolysis in more detail, beta, gamma, delta, and epsilon subunits were expressed alone in fibroblasts and their selective degradation analyzed in vitro. The beta and delta chains were degraded in the complete absence of vesicular transport, indicating their degradation in the ER membrane compartment. Proteolysis was unaffected by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), EDTA, or depletion of ATP. The gamma and epsilon subunits were stable under the same in vitro conditions, indicating that the assay reconstituted selective protein degradation within the ER. Furthermore, the results showed that the gamma and epsilon subunits did not escape degradation by being transported from the ER to pre-Golgi, or cis-Golgi, membrane compartments. Structural determinants of ER degradation contained within the membrane anchor of the TCR beta subunit were only active in permeabilized cells when reducing agents were added to the assay. Surprisingly, reducing conditions disrupted the regulation of ER proteolysis and induced rapid ER degradation of the stable CD3 gamma subunit and of a control interleukin 2 receptor chimera. Taken together, the results indicated that the ER membrane compartment regulates the selective degradation of newly synthesized proteins. Importantly, the stability of proteins retained in the ER was highly sensitive to redox conditions. It is possible that the redox buffer within the ER lumen may regulate ER protein degradation in vivo.

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.

Catalysis of acetyl-CoA cleavage and tetrahydrosarcinapterin methylation by a carbon monoxide dehydrogenase-corrinoid enzyme complex.

An enzyme complex containing carbon monoxide dehydrogenase and a corrinoid protein has been isolated from Methanosarcina barkeri. Sodium dodecyl sulfate-gel electrophoresis revealed five polypeptides of molecular masses alpha = 19,700, beta = 84,500, gamma = 63,200, delta = 53,000, and epsilon = 51,400 Da in equimolar amounts. One mol of cobamide cofactor was found per minimal alpha beta gamma delta epsilon unit. The molecular mass of the native complex was 1,600,000 Da by high pressure liquid chromatography (HPLC) gel filtration, which suggested an alpha 6 beta 6 gamma 6 delta 6 epsilon 6 oligomeric structure. Catalysis of a reaction involving cleavage of acetyl-CoA and methylation of tetrahydrosarcinapterin was indicated by spectrophotometric analyses; a time-dependent absorption decrease in the 300-320 nm region was observed in the complete reaction mixture which contained acetyl-CoA, tetrahydrosarcinapterin, and the enzyme complex. In control samples lacking any one of the these components the absorption spectrum remained virtually unaltered. Reversed-phase HPLC analysis confirmed that tetrahydrosarcinapterin was converted to a product that co-eluted with authentic methyltetrahydrosarcinapterin. The product also exhibited the UV-visible absorption spectrum expected for methyltetrahydrosarcinapterin. Free CoA was identified as an additional product of the reaction. The carbonyl group of acetyl-CoA was oxidized to carbon dioxide. Spectral changes indicated concomitant Fe/S center reduction. Production of CoA was essentially stoichiometric with methyltetrahydrosarcinapterin formation and tetrahydrosarcinapterin consumption. Analyses during purification showed that catalytic activity was restricted exclusively to the fractions that contained the carbon monoxide dehydrogenase-corrinoid enzyme complex.

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.

Six chains of the human T cell antigen receptor.CD3 complex are necessary and sufficient for processing the receptor heterodimer to the cell surface.

The T cell antigen receptor (TCR) plays a key role in the process of antigen recognition. It is a complex of at least seven peptide chains (alpha beta gamma delta epsilon zeta-zeta). It is found on the surface of mature T cells and functions in antigen binding in the presence of the major histocompatibility complex. It has been known for some time that physical associations between the CD3 proteins and the TCR chains are essential for efficient transport of either component to the surface of T cells. For example, T cells that lack either the alpha, beta, or delta chains synthesize partial complexes that are eventually degraded. cDNAs encoding the six chains of receptor have become available recently. We have used transfection techniques to generate a panel of Chinese hamster ovary cells that contain partial receptor complexes of known composition and also cells that express all six subunits of the TCR.CD3 complex. Cells in this panel were analyzed for the ability to form alpha-beta heterodimers and also an ability to transport the synthesized chains to the plasma membrane. These studies have allowed us to define the minimum requirements for TCR.CD3 expression on the cell surface.

The transmembrane anchor of the T-cell antigen receptor beta chain contains a structural determinant of pre-Golgi proteolysis.

Studies with the T-cell antigen receptor (TCR) have shown that the endoplasmic reticulum, or an organelle closely associated with it, can retain and degrade membrane proteins selectively. The observation that only three (alpha, beta, and delta) of the six (alpha beta gamma delta epsilon zeta) subunits of the TCR are susceptible to proteolysis implies that structural features within the labile proteins mark them for degradation. The TCR beta chain is degraded in the endoplasmic reticulum, and, in this study, we have started to define the domains of the protein that make it susceptible to proteolysis. The experiments show that the transmembrane anchor and short five-amino-acid cytoplasmic tail of the protein contain a dominant determinant of proteolysis. When these residues were removed from the beta chain, the protein became resistant to proteolysis. Even though the resulting ectodomain of the beta chain lacked a transmembrane anchor, it was not secreted by cells and was retained in the endoplasmic reticulum. We conclude that retention in the endoplasmic reticulum alone does not lead to degradation. The results suggest that structural features within the membrane anchor of the protein predispose the beta chain to proteolysis. This was confirmed by replacing the membrane anchor of the interleukin 2 (IL2) receptor, a protein that was stable within the secretory pathway, with that of the TCR beta chain. The unmodified IL2 receptor was transported efficiently to the surface of cells, and an "anchor minus" construct was secreted quantitatively into the culture media. When the membrane anchor of the IL2 receptor was replaced with that of the TCR beta chain, the chimera was unable to reach the Golgi apparatus and was degraded rapidly.

Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors.

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.

The implications of subunit interactions for the structure of the T cell receptor‐CD3 complex

Cell surface-expressed receptors are often multichain complexes. One of these, the T cell receptor (TcR) alpha/beta-CD3 complex, is known to contain at least seven chains: the alpha and beta TcR chains plus the gamma, delta, epsilon and two zeta chains from the CD3 complex (alpha beta gamma delta epsilon zeta 2). To gain insight into the structure of the complex we have used anti-peptide antisera specific for the individual subunits of the complex, and nonionic and ionic detergents to determine subunit interactions within the complex. Four closely associated pairs of chains could be identified: alpha beta, zeta 2, gamma epsilon and delta epsilon. Interactions between the TcR alpha beta and either gamma epsilon or delta epsilon could be observed in the apparent absence of other CD3 chains. Furthermore, a hierarchy in the strength of the association between the TcR and the individual CD3 chains could be distinguished: TcR epsilon greater than TcR delta greater than TcR gamma. The zeta 2 dimer could only be detected in "intact" TcR-CD3 complexes shedding no light on possible interactions with either the TcR or CD3-gamma, delta and epsilon chains. Finally, cross-linking experiments suggest a close spatial relationship between the TcR alpha beta and both the CD3-gamma and CD3-epsilon chains. The results demonstrate that the methods used give valuable information on subunit interactions in a cell surface-expressed receptor complex and suggest a TcR-CD3 complex in which two epsilon chains are present, one linked to gamma and the other to delta. The data further indicate that gamma epsilon and delta epsilon complexes interact directly with the TcR chains. Based on the observations a model for the structure of the TcR-CD3 is presented and discussed.

Recognition for degradation in the endoplasmic reticulum and lysosomes prevents the transport of single TCRβ and CD3δ subunits of the T-cell antigen receptor to the surface of cells

The T cell antigen receptor is a multiple subunit membrane protein made from six different polypeptide chains (alpha beta gamma delta epsilon zeta). The subunits are transmembrane proteins but only receptors assembled from all six chains are transported efficiently to the plasma membrane. Partial receptors and single subunits fail to reach the Golgi apparatus. This study has used transfected fibroblasts to follow the intracellular fate of the TCR beta and CD3 delta subunits in detail. After a lag period of approximately 90 min both chains were degraded by a process which did not require their transport to the medial Golgi. Degradation was inhibited at temperatures below 18 degrees but was unaffected by agents that disrupted the ER to Golgi transport. Experiments using transfected Chinese hamster ovary Lec 1 cells suggested that CD3 delta was degraded without transport to the cis-Golgi. Lysosomotropic agents had no effect on the proteolysis of the beta chain but did prevent the degradation of approximately 25% of the delta subunit. In the presence of chloroquine the delta subunit could be detected in lysosomes. The experiments show that proteolysis in or close to the endoplasmic reticulum plays a major role in preventing the surface expression of single beta and delta subunits of the T cell antigen receptor; nevertheless, some of the delta chain is able to evade this process. Interestingly, there is a second check on the transport of delta to the plasma membrane, and the subunit is removed from the secretory pathway and delivered to lysosomes for degradation.

Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly.

The T cell antigen receptor (TCR) is a multisubunit complex composed of at least seven transmembrane chains. The predominant species in most T cells has the composition alpha beta gamma delta epsilon zeta 2. The roles of subunit assembly in transport out of the ER and in the recently described process of pre-Golgi degradation of newly synthesized TCR chains were analyzed in a T-cell line deficient in the synthesis of delta chains (delta 2) and in COS-1 fibroblasts transfected with genes encoding individual TCR chains. Studies with the delta-deficient T-cell line showed that, in the absence of delta, the other TCR chains were synthesized at normal rates, but, instead of being transported to the cell surface, they were retained in the ER. Analysis of the fate of TCR chains retained in the ER showed that they were degraded at vastly different rates by a nonlysosomal pathway. Whereas the alpha chains were degraded rapidly, gamma, zeta, and epsilon were relatively long-lived. To analyze whether this selective degradation was because of different intrinsic susceptibility of the individual chains to degradation or to the formation of resistant oligomers, TCR chains were expressed alone or in combinations in COS-1 fibroblasts. These studies showed that (a) individual TCR chains were degraded at different rates when expressed alone in COS-1 cells, and (b) sensitive chains could be stabilized by coexpression with a resistant chain. Taken together, these observations indicate that both intrinsic sensitivity and subunit assembly play a role in determining the rates at which newly synthesized TCR chains are degraded in the ER.

Subunit interactions within the T-cell antigen receptor: clues from the study of partial complexes.

The T-cell antigen receptor is a multisubunit complex composed of seven transmembrane chains (alpha beta gamma delta epsilon zeta 2). Subunit interactions within this complex were defined by analyzing the subunit composition of partial complexes. These partial complexes were observed in mutant and tumor T cells that fail to synthesize one or more of the receptor chains or in fibroblasts transfected with genes encoding T-cell antigen receptor chains. In addition, partial complexes were generated by immunoprecipitation with antibodies that cause selective dissociation of T-cell antigen receptor chains. The alpha and beta chains were found to form a disulfide-linked dimer in the absence of any of the other chains. The gamma, delta, and epsilon chains were also efficiently associated in the absence of a complete heterodimer. Complexes composed only of delta epsilon or gamma epsilon could be observed. Both these dimers, as well as the gamma delta epsilon trimer, could form stable complexes with alpha beta, even in the absence of zeta 2. The zeta 2 dimer could bind directly to alpha beta. In the absence of a complete clonotypic heterodimer, zeta 2 was not stably associated with gamma delta epsilon. These observations suggest a model in which alpha beta interacts directly with the gamma delta epsilon trimer and zeta 2, with less-direct interaction between the latter two.