scholarly journals Biogenesis of membrane-bound and secreted immunoglobulins. I. Two distinct translation products of human mu-chain, with identical N-termini and different C-termini.

1980 ◽  
Vol 152 (2) ◽  
pp. 463-468 ◽  
Author(s):  
J M McCune ◽  
V R Lingappa ◽  
S M Fu ◽  
G Blobel ◽  
H G Kunkel
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Posttranslational Modifications ◽  
Messenger Rna ◽  
Free System ◽  
Human Lymphoblastoid Cell ◽  
C Terminus ◽  
Molecular Weight Difference ◽  
Membrane Bound

Structural differences between the heavy chain of membrane-bound IgM (mu m) and the heavy chain of secreted IgM (mu s) were investigated. The primary translation products of the mu-chain, free of posttranslational modifications, were synthesized in a wheat-germ cell-free system, programmed with messenger RNA derived from human lymphoblastoid cell lines positive for both membrane-bound and secreted IgM. Encoded in this sytem were two mu-chains, which shared N-terminal signal peptides and which differed both in molecular weight and in C-terminal amino acid sequence. In vivo pulse labeling of cells confirmed that, as intermediates in the rough endoplasmic reticulum, these two forms expressed the same idiotype and maintained their difference in molecular weight and in C-terminal sequence. By correlation with pulse-chase kinetics and with immunofluorescence, one form of mu-chain represents mu m, and the other, mu s. Because the molecular weight difference between the two is manifest at the level of their primary translation products, these studies demonstrate that mu m is distinguished from mu s by a difference in primary structure, at least in part at the C-terminus.

scholarly journals Biogenesis of membrane-bound and secreted immunoglobulins. II. Two forms of the human alpha chain translated in vitro and processed in vivo as distinct polypeptide chains.

1981 ◽  
Vol 153 (6) ◽  
pp. 1684-1689 ◽  
Author(s):  
J M McCune ◽  
S M Fu ◽  
G Blobel ◽  
H G Kunkel
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Messenger Rna ◽  
Secretory Iga ◽  
Free System ◽  
Alpha Chain ◽  
Functional Consequences ◽  
Polypeptide Chains

Structural differences between alpha m (ther heavy chain of membrane IgA) and alpha s (the heavy chain of secretory IgA) were investigated. Messenger RNA from the human B lymphoblastoid line 32a.1, expressing both membrane and secretory IgA, was translated in a wheat germ cell-free system, resulting in the synthesis of two primary translation products for the alpha chain, that differed in molecular weight. In vivo pulse and pulse-chase experiments demonstrated that two early biosynthetic forms of the alpha chain were subsequently modified to yield three intracellular forms. As shown by endo-beta-N-acetylglucosaminidase H (endo H) treatment, these forms represent two alpha polypeptide chains, with varying compositions of N-linked oligosaccharides. Of the two forms of the alpha chain remaining after endo H treatment, only the form with the lowest molecular weight was associated with cells after long chase periods. The possible significance of this difference from the results with mu and delta chains is discussed. These results indicate that alpha m is distinguished from an alpha s by a difference in both primary structure and intracellular processing. The functional consequences of this distinction, previously shown for the heavy chain of membrane IgM (micrometer) and heavy chain of secretory IgM (microseconds), may reflect a principle common to the secretory and membrane forms of all immunoglobulin heavy chain classes.

scholarly journals Effect of post-ischaemic recovery on albumin synthesis and relative amount of translatable albumin messenger RNA in rat liver

1982 ◽  
Vol 204 (1) ◽  
pp. 197-202 ◽  
Author(s):  
G Cairo ◽  
L Schiaffonati ◽  
M G Aletti ◽  
A Bernelli-Zazzera
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Messenger Rna ◽  
Relative Proportion ◽  
Liver Homogenate ◽  
Albumin Synthesis ◽  
Specific Proteins ◽  
Membrane Bound ◽  
Albumin Mrna

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.

scholarly journals Allelic exclusion in transgenic mice carrying mutant human IgM genes.

1988 ◽  
Vol 167 (6) ◽  
pp. 1969-1974 ◽  
Author(s):  
M C Nussenzweig ◽  
A C Shaw ◽  
E Sinn ◽  
J Campos-Torres ◽  
P Leder
Keyword(s):  
Transgenic Mice ◽  
Heavy Chain ◽  
Chain Gene ◽  
Allelic Exclusion ◽  
Heavy Chain Gene ◽  
F1 Hybrid ◽  
Membrane Bound ◽  
Ig Heavy Chain ◽  
Simultaneous Expression

Expression of the membrane-bound version of the human mu chain in transgenic mice results in the allelic exclusion of endogenous mouse Ig heavy chain genes (6). The secreted version of the human Ig transgene has no such effect. F1 hybrid animals that carry transgenes for both secreted and membrane-bound human mu chains produce both forms of the human heavy chain while strongly suppressing endogenous mouse mu expression. The simultaneous expression of the two rearranged transgenes in primary B cells suggests that allelic exclusion operates before the formation of a second functionally rearranged heavy chain gene in vivo.

scholarly journals Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

1997 ◽  
Vol 8 (4) ◽  
pp. 621-636 ◽  
Author(s):  
J M Caron
Keyword(s):  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Hydrophobic Interactions ◽  
Covalent Attachment ◽  
Protein Structure Analysis ◽  
Free System ◽  
Intracellular Membranes ◽  
Cell Free System ◽  
Cytoplasmic Proteins

It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

scholarly journals Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

2021 ◽  
pp. mbc.E20-07-0500
Author(s):  
Eri Hirata ◽  
Kyo Shirai ◽  
Tatsuya Kawaoka ◽  
Kosuke Sato ◽  
Fumito Kodama ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Multivesicular Body ◽  
Catalytic Triad ◽  
Double Membrane ◽  
Terminal Domain ◽  
C Terminus ◽  
N Terminus ◽  
Membrane Bound ◽  
Important Residue

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of both domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad, and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to understanding how the C-terminal lipase domain recognizes autophagic bodies to degrade them. [Media: see text] [Media: see text]

scholarly journals The eucaryotic aminoacyl-tRNA synthetase complex: suggestions for its structure and function.

1984 ◽  
Vol 99 (2) ◽  
pp. 373-377 ◽  
Author(s):  
M P Deutscher
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Structural Similarity ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Membrane Bound ◽  
And Function ◽  
Molecular Weight Form

Aminoacyl-tRNA synthetases from eucaryotic cells generally are isolated as high molecular weight complexes comprised of multiple synthetase activities, and often containing other components as well. A model is proposed for the synthetase complex in which hydrophobic extensions on the proteins serve to maintain them in their high molecular weight form, but are not needed for catalytic activity. The structural similarity of these enzymes to certain membrane-bound proteins, and its implications for synthetase localization and function in vivo, are discussed.

scholarly journals Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the α-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the β-subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 659-665 ◽  
Author(s):  
David C. A. JOHN ◽  
Neil J. BULLEID
Keyword(s):  
Heavy Chain ◽  
Binding Protein ◽  
Immunoglobulin Heavy Chain ◽  
Β Subunit ◽  
Intact Cells ◽  
Α Subunit ◽  
Free System ◽  
A Cell ◽  
Α Subunits

Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an α2β2 tetramer from newly synthesized α-subunits associating with an endogenous pool of β-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled α-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018–14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized α-subunits were shown to assemble with the β-subunit, to form insoluble aggregates, or to remain soluble but not associate with the β-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and α-subunits were co-immunoprecipitated with antibodies against the α-subunit and BiP, respectively. These results indicate that unassembled α-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.

scholarly journals C-terminal modification of the insulin B:11–23 peptide creates superagonists in mouse and human type 1 diabetes

2017 ◽  
Vol 115 (1) ◽  
pp. 162-167 ◽  
Author(s):  
Yang Wang ◽  
Tomasz Sosinowski ◽  
Andrey Novikov ◽  
Frances Crawford ◽  
David B. Neau ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
T Cell Activation ◽  
Posttranslational Modifications ◽  
Cell Activation ◽  
Nod Mice ◽  
Side Chain ◽  
C Terminus

A polymorphism at β57 in some major histocompatibility complex class II (MHCII) alleles of rodents and humans is associated with a high risk for developing type 1 diabetes (T1D). However, a highly diabetogenic insulin B chain epitope within the B:9–23 peptide is presented poorly by these alleles to a variety of mouse and human CD4 T cells isolated from either nonobese diabetic (NOD) mice or humans with T1D. We have shown for both species that mutations at the C-terminal end of this epitope dramatically improve presentation to these T cells. Here we present the crystal structures of these mutated peptides bound to mouse IAg7 and human HLA-DQ8 that show how the mutations function to improve T-cell activation. In both peptide binding grooves, the mutation of B:22R to E in the peptide changes a highly unfavorable side chain for the p9 pocket to an optimal one that is dependent on the β57 polymorphism, accounting for why these peptides bind much better to these MHCIIs. Furthermore, a second mutation of the adjacent B:21 (E to G) removes a side chain from the surface of the complex that is highly unfavorable for a subset of NOD mouse CD4 cells, thereby greatly enhancing their response to the complex. These results point out the similarities between the mouse and human responses to this B chain epitope in T1D and suggest there may be common posttranslational modifications at the C terminus of the peptide in vivo to create the pathogenic epitopes in both species.

scholarly journals Control of immunoglobulin secretion in the murine plasmacytoma line MOPC 315.

1978 ◽  
Vol 148 (1) ◽  
pp. 301-312 ◽  
Author(s):  
G E Sonenshein ◽  
M Siekevitz ◽  
G R Siebert ◽  
M L Gefter
Keyword(s):  
Heavy Chain ◽  
Wheat Germ ◽  
Secretory Protein ◽  
Synthesis Rate ◽  
Free System ◽  
Alpha Chain ◽  
Chain Synthesis ◽  
Variant Line

Cells of the 315LV-1 (derived from NP1) variant line of MOPC 315 contain approximately 1% the normal intracellular level of the heavy (alpha) chain of IgA and no detectable light (lambda2) chain. The synthesis rate of alpha-chain in the variant, however, is similar to that in cells of the parent line. Moreover the relative amount of translatable alpha-chain mRNA that can be extracted from 315LV-1 cells is about the same as for parental cells. No light-chain synthesis can be detected either in vivo or in vitro in a wheat germ cell-free system. The 315LV-1 heavy chain synthesized in vivo or in vitro has slightly greater electrophoretic mobility than normal H chain and turns over rapidly intracellularly. The variant fails to secrete any of its heavy chain, despite the fact that its H chain mRNA is bound to membranes, as one would expect for a secretory protein message. Fusion of 315LV-1 cells with cells of a kappa-producing MPC 11 variant line leads to stabilization of the intracellular H chain and also to full recovery of secretion of the H chain as an H2L2 molecule.

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