scholarly journals Identification and subsequent phosphorylation of sequestered partially processed caseins in the lactating guinea-pig mammary gland

1984 ◽  
Vol 222 (2) ◽  
pp. 501-510 ◽  
Author(s):  
A P Boulton ◽  
J C Pascall ◽  
R K Craig
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Secretory Pathway ◽  
Short Pulse ◽  
Early Event ◽  
Post Translational Modification ◽  
Electrophoretic Mobilities ◽  
Membrane Bound

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.

scholarly journals Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland

1979 ◽  
Vol 181 (3) ◽  
pp. 737-756 ◽  
Author(s):  
R K Craig ◽  
A P Boulton ◽  
O S Harrison ◽  
D Parker ◽  
P N Campbell
Keyword(s):  
Protein Synthesis ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Complexity Analysis ◽  
Free Protein ◽  
Mrna Species ◽  
Run Off ◽  
Membrane Bound ◽  
Cell Free Protein Synthesis

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87–93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

scholarly journals Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland

1980 ◽  
Vol 192 (2) ◽  
pp. 489-498 ◽  
Author(s):  
Ian C. Bathurst ◽  
Roger K. Craig ◽  
David G. Herries ◽  
Peter N. Campbell
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Regulation Of Gene Expression ◽  
Milk Proteins ◽  
Relative Distribution ◽  
Free System ◽  
Low Salt ◽  
Nuclear Rna ◽  
System 2

1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)–cellulose into three populations; those that bound at ‘low salt’ [long poly(A) tracts, 78–32 nucleotides]; those that bound at ‘high salt’ [shorter poly(A) tracts, 48–21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ‘low salt’ and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. 3H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ‘low-salt’ post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ‘low-salt’ poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6×105 copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ‘abundant’ and so-called ‘moderately abundant’ sequences were present in essentially identical concentrations (2×103 copies of each sequence/cell). The majority of these (90–95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.

scholarly journals Uptake and metabolism of tryptophan by the isolated perfused guinea-pig mammary gland

1976 ◽  
Vol 158 (3) ◽  
pp. 659-662 ◽  
Author(s):  
B Mepham ◽  
A R Peters ◽  
S R Davis
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Blood Plasma ◽  
Milk Protein ◽  
Absorption Studies ◽  
Uptake And Metabolism ◽  
Tryptophan Uptake

Tryptophan uptake by the isolated perfused lactating guinea-pig mammary gland was 46.5+/-4.6 mug/h per g. Results of absorption studies and the use of [methylene-14C]tryptophan suggest that tryptophan is one of the group of amino acids that are transferred almost quantitatively from blood plasma to milk protein.

scholarly journals GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Hung-Hsiang Huang ◽  
Antti Hassinen ◽  
Subha Sundaram ◽  
Andrej-Nikolai Spiess ◽  
Sakari Kellokumpu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Inhibitory Activity ◽  
Secretory Pathway ◽  
Cultured Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bimolecular Fluorescence Complementation ◽  
Invariant Chain ◽  
Fluorescent Resonance Energy Transfer ◽  
Membrane Bound

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.

scholarly journals Life Stage-Specific Cargo Receptors Facilitate Glycosylphosphatidylinositol-Anchored Surface Coat Protein Transport in Trypanosoma brucei

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Emilia K. Kruzel ◽  
George P. Zimmett ◽  
James D. Bangs
Keyword(s):  
Endoplasmic Reticulum ◽  
Virulence Factor ◽  
Secretory Pathway ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Host Immune System ◽  
Protozoan Parasites ◽  
Gpi Anchor ◽  
African Trypanosomes ◽  
Membrane Bound

ABSTRACT African trypanosomes are protozoan parasites that cause African sleeping sickness. Critical to the success of the parasite is the variant surface glycoprotein (VSG), which covers the parasite cell surface and which is essential for evasion of the host immune system. VSG is membrane bound by a glycolipid (GPI) anchor that is attached in the earliest compartment of the secretory pathway, the endoplasmic reticulum (ER). We have previously shown that the anchor acts as a positive forward trafficking signal for ER exit, implying a cognate receptor mechanism for GPI recognition and loading in coated cargo vesicles leaving the ER. Here, we characterize a family of small transmembrane proteins that act at adaptors for this process. This work adds to our understanding of general GPI function in eukaryotic cells and specifically in the synthesis and transport of the critical virulence factor of pathogenic African trypanosomes. The critical virulence factor of bloodstream-form Trypanosoma brucei is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). Endoplasmic reticulum (ER) exit of VSG is GPI dependent and relies on a discrete subset of COPII machinery (TbSec23.2/TbSec24.1). In other systems, p24 transmembrane adaptor proteins selectively recruit GPI-anchored cargo into nascent COPII vesicles. Trypanosomes have eight putative p24s (TbERP1 to TbERP8) that are constitutively expressed at the mRNA level. However, only four TbERP proteins (TbERP1, -2, -3, and -8) are detectable in bloodstream-form parasites. All four colocalize to ER exit sites, are required for efficient GPI-dependent ER exit, and are interdependent for steady-state stability. These results suggest shared function as an oligomeric ER GPI-cargo receptor. This cohort also mediates rapid forward trafficking of the soluble lysosomal hydrolase TbCatL. Procyclic insect-stage trypanosomes have a distinct surface protein, procyclin, bearing a different GPI anchor structure. A separate cohort of TbERP proteins (TbERP1, -2, -4, and -8) are expressed in procyclic parasites and also function in GPI-dependent ER exit. Collectively, these results suggest developmentally regulated TbERP cohorts, likely in obligate assemblies, that may recognize stage-specific GPI anchors to facilitate GPI-cargo trafficking throughout the parasite life cycle. IMPORTANCE African trypanosomes are protozoan parasites that cause African sleeping sickness. Critical to the success of the parasite is the variant surface glycoprotein (VSG), which covers the parasite cell surface and which is essential for evasion of the host immune system. VSG is membrane bound by a glycolipid (GPI) anchor that is attached in the earliest compartment of the secretory pathway, the endoplasmic reticulum (ER). We have previously shown that the anchor acts as a positive forward trafficking signal for ER exit, implying a cognate receptor mechanism for GPI recognition and loading in coated cargo vesicles leaving the ER. Here, we characterize a family of small transmembrane proteins that act at adaptors for this process. This work adds to our understanding of general GPI function in eukaryotic cells and specifically in the synthesis and transport of the critical virulence factor of pathogenic African trypanosomes.

scholarly journals Novel Aspects of Degradation of T Cell Receptor Subunits from the Endoplasmic Reticulum (ER) in T Cells: Importance of Oligosaccharide Processing, Ubiquitination, and Proteasome-dependent Removal from ER Membranes

1998 ◽  
Vol 187 (6) ◽  
pp. 835-846 ◽  
Author(s):  
Mei Yang ◽  
Satoshi Omura ◽  
Juan S. Bonifacino ◽  
Allan M. Weissman
Keyword(s):  
T Cells ◽  
Endoplasmic Reticulum ◽  
T Cell ◽  
Secretory Pathway ◽  
Cell Receptor ◽  
Dependent Manner ◽  
Oligosaccharide Processing ◽  
Membrane Bound ◽  
Α Subunits ◽  
Er Membrane

Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane. TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal processes that have been referred to as “ER degradation”. The molecular basis for the loss of the TCR CD3-δ and TCR-α subunits from the ER was investigated in lymphocytes. For CD3-δ, we describe a process leading to its degradation that includes trimming of mannose residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of CD3-δ was markedly curtailed and CD3-δ remained membrane bound in a complex with CD3-ε. TCR-α was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-α, and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target proteins from the ER to the cytosol for degradation.

scholarly journals The ribonucleic acids of the mammary gland of the guinea pig

1975 ◽  
Vol 146 (3) ◽  
pp. 575-583
Author(s):  
P Ashby ◽  
P N Campbell
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Gel Electrophoresis ◽  
Milk Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mammary Glands ◽  
Ribosomal Subunits ◽  
Ribonucleic Acids ◽  
Lactating Mammary Gland ◽  
Zone Velocity

1. 32-P-labelled polyribosome preparations were made from the mammary glands of lactating and late-pregnant guinea pigs after injection of (32-P)i into the animals. 2. The RNA of polyribosomes, ribosomal subunits and that released from polyribosomes by EDTA were analysed by zone velocity centrifugation and by polyacrylamide-gel electrophoresis. 3. RNA species which have the physical properties expected for the milk protein mRNA were detected. RNA species of a size which could code for the caseins were present in lactating but not in pre-lactating mammary-gland polyribosomes.

scholarly journals How Viruses Hijack and Modify the Secretory Transport Pathway

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2535
Author(s):  
Zubaida Hassan ◽  
Nilima Dinesh Kumar ◽  
Fulvio Reggiori ◽  
Gulfaraz Khan
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Eukaryotic Cells ◽  
Environmental Cues ◽  
Transport Pathway ◽  
Dynamic Membrane ◽  
Enveloped Viruses ◽  
Vesicular Traffic ◽  
Secretory Transport ◽  
Membrane Bound

Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae, Poxviridae, Parvoviridae and Herpesviridae families.

scholarly journals Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation

2000 ◽  
Vol 348 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Armando J. PARODI
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Cell Recognition ◽  
Glucosidase Ii ◽  
Membrane Bound ◽  
Quaternary Structures ◽  
Lectin Recognition

The endoplasmic reticulum (ER) is the subcellular site where proteins following the secretory pathway acquire their proper tertiary and, in certain cases, quaternary structures. Species that are not yet properly folded are prevented from exit to the Golgi apparatus and, if permanently misfolded, are transported to the cytosol, where they are degraded in the proteasomes. This review deals with a mechanism, applicable to proteins that are N-glycosylated in the ER, by which the quality control of folding is performed. Protein-linked monoglucosylated glycans, formed by glucosidase I- and glucosidase II-dependent partial deglucosylation of the oligosaccharides transferred from dolichol diphosphate derivatives in N-glycosylation (Glc3Man9GlcNAc2), mediate glycoprotein recognition by two ER-resident lectins, membrane-bound calnexin (CNX) and its soluble homologue, calreticulin (CRT). A still not yet fully confirmed interaction between the lectins and the protein moieties of folding glycoproteins may occur after lectin recognition of monoglucosylated structures. Further deglucosylation of glycans by glucosidase II, and perhaps also by a change in CNX/CRT and/or in the substrate glycoprotein conformation, liberates the glycoproteins from their CNX/CRT anchors. Glycans may be then reglucosylated by the UDP-Glc:glycoprotein glucosyltransferase (GT), and thus be recognized again by CNX/CRT, but only when linked to not yet properly folded protein moieties, as this enzyme behaves as a sensor of glycoprotein conformation. Deglucosylation/reglucosylation cycles catalysed by the opposing activities of glucosidase II and GT only stop when proper folding is achieved. The interaction between CNX/CRT and a monoglucosylated glycan is one of the alternative mechanisms by which cells retain not yet properly folded glycoproteins in the ER; in addition, it enhances folding efficiency by preventing protein aggregation and thus allowing intervention of classical chaperones and other folding-assisting proteins. There is evidence suggesting that both glycoprotein glucosylation and mannose removal, respectively mediated by GT and ER mannosidase I, might be involved in cell recognition of permanently misfolded glycoproteins bound for proteasome degradation.

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