The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi.

Dithiothreitol (DTT) blocks the endoplasmic reticulum (ER)-Golgi transport of newly synthesized immunoglobulin (Ig) molecules, whereas 2-mercaptoethanol (2ME) allows secretion of unpolymerized Igs otherwise retained intracellularly by disulphide interchange reactions. To understand this dichotomy, we have compared the effects of DTT and 2ME on the assembly, intracellular transport, and secretion of a panel of chimeric Igs that are either constitutively secreted or retained intracellularly. Our results demonstrate that DTT, but not 2ME, reduces some of the inter- and intrachain disulphide bonds and causes partial disassembly of H2L2 complexes and unfolding of individual chains in the ER. Upon DTT removal, heavy (H) and light (L) chains reform hapten-binding H2L2 molecules, which are later secreted. Reduction of the H2L2 interchain disulphide bonds can occur along the entire secretory pathway; however, in or beyond the Golgi this does not result in efficient H-L disassembly or unfolding. As a consequence, DTT does not block the exit from the Golgi. Moreover, unpolymerized Igs--normally retained in a pre-Golgi compartment--no longer require reducing agents to be secreted once they have reached the Golgi. Thus, little if any thiol-mediated retention seems to take place in or beyond the Golgi complex.

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Potassium depletion inhibits the intracellular transport of secretory proteins between the endoplasmic reticulum and the Golgi complex

Journal of Cell Science ◽

10.1242/jcs.92.2.173 ◽

1989 ◽

Vol 92 (2) ◽

pp. 173-185

Author(s):

J.D. Judah ◽

K.E. Howell ◽

J.A. Taylor ◽

P.S. Quinn

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Intracellular Transport ◽

Secretory Pathway ◽

Subcellular Fractionation ◽

Secretory Proteins ◽

Mature Form ◽

Processing Enzymes ◽

Microscopic Studies ◽

K Depletion

In this paper we show that hepatocytes that have been depleted of K+ secrete albumin, alpha-1-anti-trypsin and transferrin at a slower rate than cells to which K+ has been returned. K+ depletion has no effect on the intracellular nucleotide pools, and we provide evidence that the inhibitions of secretion caused by depletion of K+ and depletion of ATP are independent. Studies of the processing of alpha-1-anti-trypsin show that K+ depletion inhibits the formation of the mature form of the protein, but that immature forms are never secreted. In cells to which K+ was returned, secretion of the mature form was restored. This implies that transport is blocked at a point before the proteins reach the processing enzymes. Proteins delayed by K+ depletion are not removed from the secretory pathway, but are free to mix with protein synthesized subsequently. These data are supported by subcellular fractionation experiments, which show that the secretory proteins are delayed before reaching the Golgi complex, and by immunoelectron microscopic studies. These show that in K+-deficient cells the morphology of both the endoplasmic reticulum and the Golgi complex is normal. The secretory proteins are trapped in smooth vesicles that contain reaction product when incubated for glucose-6-phosphatase, a marker for the endoplasmic reticulum.

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Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

The Journal of Cell Biology ◽

10.1083/jcb.103.4.1179 ◽

1986 ◽

Vol 103 (4) ◽

pp. 1179-1191 ◽

Cited By ~ 243

Author(s):

C S Copeland ◽

R W Doms ◽

E M Bolzau ◽

R G Webster ◽

A Helenius

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Complex ◽

Intracellular Transport ◽

Secretory Pathway ◽

Quaternary Structure ◽

Subunit Interactions ◽

Oligomer Formation ◽

Influenza Hemagglutinin ◽

Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

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Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

Biochemical Society Symposium ◽

10.1042/bss2007c17 ◽

2007 ◽

Vol 74 ◽

pp. 199-209 ◽

Cited By ~ 3

Author(s):

Régine Bosson ◽

Andreas Conzelmann

Keyword(s):

Fatty Acids ◽

Endoplasmic Reticulum ◽

Fatty Acid ◽

Intracellular Transport ◽

Secretory Pathway ◽

Gpi Anchor ◽

New Class ◽

Golgi Transport ◽

Lipid Moiety ◽

Gpi Proteins

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C26 fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C16 and C18 fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C26 fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C26 in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor.

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The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.2980-2993.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 2980-2993

Author(s):

R Ossig ◽

C Dascher ◽

H H Trepte ◽

H D Schmitt ◽

D Gallwitz

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Protein Transport ◽

Single Copy ◽

Integral Membrane Protein ◽

Carboxypeptidase Y ◽

Null Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

Golgi Transport ◽

Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

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Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

The Journal of Cell Biology ◽

10.1083/jcb.115.1.31 ◽

1991 ◽

Vol 115 (1) ◽

pp. 31-43 ◽

Cited By ~ 235

Author(s):

H Plutner ◽

A D Cox ◽

S Pind ◽

R Khosravi-Far ◽

J R Bourne ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Essential Role ◽

Secretory Pathway ◽

Binding Protein ◽

Vesicular Transport ◽

Initial Step ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

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New Mutants of Saccharomyces cerevisiae Affected in the Transport of Proteins From the Endoplasmic Reticulum to the Golgi Complex

Genetics ◽

10.1093/genetics/142.2.393 ◽

1996 ◽

Vol 142 (2) ◽

pp. 393-406 ◽

Cited By ~ 11

Author(s):

Linda J Wuestehube ◽

Rainer Duden ◽

Arlene Eun ◽

Susan Hamamoto ◽

Paul Korn ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Secretory Protein ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Membrane Structures ◽

Α Subunit ◽

Complementation Groups ◽

Vacuolar Protein

Abstract We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.

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Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

Journal of Cell Science ◽

10.1242/jcs.92.4.633 ◽

1989 ◽

Vol 92 (4) ◽

pp. 633-642

Author(s):

J.K. Burkhardt ◽

Y. Argon

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Late Stage ◽

Intracellular Transport ◽

Post Translational Modifications ◽

Glycoprotein G ◽

Golgi Compartment ◽

Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

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Pathogenic Effects of Impaired Retrieval between the Endoplasmic Reticulum and Golgi Complex

International Journal of Molecular Sciences ◽

10.3390/ijms20225614 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5614 ◽

Cited By ~ 6

Author(s):

Hiroshi Kokubun ◽

Hisayo Jin ◽

Tomohiko Aoe

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Protein Transport ◽

Receptor Activation ◽

Toxic Substances ◽

Bidirectional Transport ◽

The Unfolded Protein Response ◽

Kdel Receptor ◽

Kdel Sequence

Cellular activities, such as growth and secretion, are dependent on correct protein folding and intracellular protein transport. Injury, like ischemia, malnutrition, and invasion of toxic substances, affect the folding environment in the endoplasmic reticulum (ER). The ER senses this information, following which cells adapt their response to varied situations through the unfolded protein response. Activation of the KDEL receptor, resulting from the secretion from the ER of chaperones containing the KDEL sequence, plays an important role in this adaptation. The KDEL receptor was initially shown to be necessary for the retention of KDEL sequence-containing proteins in the ER. However, it has become clear that the activated KDEL receptor also regulates bidirectional transport between the ER and the Golgi complex, as well as from the Golgi to the secretory pathway. In addition, it has been suggested that the signal for KDEL receptor activation may also affect several other cellular activities. In this review, we discuss KDEL receptor-mediated bidirectional transport and signaling and describe disease models and human diseases related to KDEL receptor dysfunction.

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The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

Biochemical Journal ◽

10.1042/bj2370033 ◽

1986 ◽

Vol 237 (1) ◽

pp. 33-39 ◽

Cited By ~ 31

Author(s):

E Fries ◽

I Lindström

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Low Temperatures ◽

Intracellular Transport ◽

Rat Hepatocytes ◽

Subcellular Fractionation ◽

Isolated Rat Hepatocytes ◽

Different Temperatures ◽

Lag Period ◽

Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

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Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.10.4.1043 ◽

1999 ◽

Vol 10 (4) ◽

pp. 1043-1059 ◽

Cited By ~ 82

Author(s):

Wolfgang P. Barz ◽

Peter Walter

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Secretory Pathway ◽

Protein Transport ◽

Protein Translocation ◽

Sequence Similarity ◽

Surface Proteins ◽

Golgi Transport ◽

Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

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