scholarly journals Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus.

1994 ◽  
Vol 126 (1) ◽  
pp. 41-52 ◽  
Author(s):  
C Hammond ◽  
A Helenius
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Gradient Centrifugation ◽  
Membrane Glycoprotein ◽  
Folding Defect ◽  
Selective Retention ◽  
Vesicular Stomatitis ◽  
Intermediate Compartment ◽  
Golgi Network

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and assembled conformation. To analyze how the selective retention of misfolded proteins works, we monitored the long-term fate of a membrane glycoprotein with a temperature-dependent folding defect, the G protein of tsO45 vesicular stomatitis virus. We used indirect immunofluorescence, immunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and the Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF4-, and coimmunoprecipitation with calnexin antibodies. The results showed that the misfolded G protein is not retained in the ER alone; it can move to the intermediate compartment and to the cis-Golgi network but is then recycled back to the ER. In the ER it is associated with calnexin and BiP/GRP78. Of these two chaperones, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of multiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.

scholarly journals Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex.

1992 ◽  
Vol 118 (1) ◽  
pp. 43-50 ◽  
Author(s):  
L V Lotti ◽  
M R Torrisi ◽  
M C Pascale ◽  
S Bonatti
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Time Course ◽  
Secretory Pathway ◽  
Vero Cells ◽  
Protein G ◽  
Marker Protein ◽  
Immunocytochemical Analysis ◽  
Vesicular Stomatitis ◽  
Intermediate Compartment

We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.

scholarly journals Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.

1987 ◽  
Vol 105 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R W Doms ◽  
D S Keller ◽  
A Helenius ◽  
W E Balch
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Gradient Centrifugation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Protein Aggregate ◽  
Vesicular Stomatitis

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.

scholarly journals gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

1994 ◽  
Vol 124 (5) ◽  
pp. 649-665 ◽  
Author(s):  
J Alcalde ◽  
G Egea ◽  
IV Sandoval
Keyword(s):  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Low Temperature ◽  
Golgi Complex ◽  
Low Temperatures ◽  
Membrane Glycoprotein ◽  
Golgi Membranes ◽  
Intermediate Compartment ◽  
Golgi Network ◽  
In Cells

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.

scholarly journals Involvement of the Transmembrane Protein p23 in Biosynthetic Protein Transport

1997 ◽  
Vol 139 (5) ◽  
pp. 1119-1135 ◽  
Author(s):  
Manuel Rojo ◽  
Rainer Pepperkok ◽  
Gregory Emery ◽  
Roland Kellner ◽  
Espen Stang ◽  
...  
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Transmembrane Protein ◽  
Membrane Surface ◽  
Membrane Recruitment ◽  
Intermediate Compartment ◽  
Golgi Network

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/μm2 membrane surface area, or ≈30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment . Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

scholarly journals Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

2012 ◽  
Vol 23 (12) ◽  
pp. 2339-2351 ◽  
Author(s):  
Yogikala Prabhu ◽  
Patricia V. Burgos ◽  
Christina Schindler ◽  
Ginny G. Farías ◽  
Javier G. Magadán ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Amyloid Precursor Protein Processing ◽  
Intermediate Compartment ◽  
Golgi Network

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.

Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules

1997 ◽  
Vol 3 (S2) ◽  
pp. 139-140
Author(s):  
John Presley ◽  
Koret Hirschberg ◽  
Nelson Cole
Keyword(s):  
Green Fluorescent Protein ◽  
Membrane Transport ◽  
G Protein ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dynamic Properties ◽  
Living Cells ◽  
Morphological Properties ◽  
Green Fluorescent

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

scholarly journals The dynamic nature of the Golgi complex.

1989 ◽  
Vol 108 (2) ◽  
pp. 277-297 ◽  
Author(s):  
G Griffiths ◽  
S D Fuller ◽  
R Back ◽  
M Hollinshead ◽  
S Pfeiffer ◽  
...  
Keyword(s):  
Surface Area ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Total Surface Area ◽  
Morphological Evidence ◽  
Golgi Stack ◽  
Golgi Compartment ◽  
Vesicular Stomatitis ◽  
Golgi Network

The intracellular transport of newly synthesized G protein of vesicular stomatitis virus is blocked at 20 degrees C and this spanning membrane glycoprotein accumulates in the last Golgi compartment, the trans Golgi-network (TGN). Previous morphological evidence suggested that the TGN enlarged significantly under this condition. In the present study we have used stereological procedures to estimate the volume and surface area of the Golgi stack and the TGN of baby hamster kidney cells under different conditions. The results indicate that the increase in the size of the TGN at 20 degrees C is accompanied by a significant decrease in the surface area and volume of the preceding Golgi compartments. A similar effect is also seen in uninfected cells at 20 degrees C, as well as during normal (37 degrees C) infection with Semliki Forest virus. In the latter case, however, the decrease in the size of the Golgi stack and the increase in that of the TGN is not accompanied by inhibition of transport from the Golgi complex to the cell surface. The results indicate that the Golgi stack and the TGN are dynamic and interrelated structures that are capable of rapid alteration in total surface area in response to changes in the rates of membrane transport.

scholarly journals KDEL and KKXX Retrieval Signals Appended to the Same Reporter Protein Determine Different Trafficking between Endoplasmic Reticulum, Intermediate Compartment, and Golgi Complex

2003 ◽  
Vol 14 (3) ◽  
pp. 889-902 ◽  
Author(s):  
Mariano Stornaiuolo ◽  
Lavinia V. Lotti ◽  
Nica Borgese ◽  
Maria-Rosaria Torrisi ◽  
Giovanna Mottola ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Reporter Protein ◽  
Transport Vesicles ◽  
Efficient Retrieval ◽  
Intermediate Compartment ◽  
Almost All

Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, whereO-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms.

scholarly journals ER to Golgi Transport

1999 ◽  
Vol 147 (6) ◽  
pp. 1205-1222 ◽  
Author(s):  
Cecilia Alvarez ◽  
Hideaki Fujita ◽  
Ann Hubbard ◽  
Elizabeth Sztul
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Intact Cell ◽  
Cell Transport ◽  
Golgi Stack ◽  
Golgi Transport ◽  
Transport Assay ◽  
Golgi Structure

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti–p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca2+-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15°C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

Golgi-bound Rab34 is a novel member of the secretory pathways

2007 ◽  
Vol 30 (4) ◽  
pp. 82
Author(s):  
Neil M. Goldenberg ◽  
Sergio Grinstein ◽  
Mel Silverman
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Class I Mhc ◽  
Golgi Stack ◽  
Vesicular Stomatitis ◽  
Golgi Network ◽  
Intracellular Vesicle Transport

Background: Golgi-localized Rab34 has been implicated in repositioning of lysosomes and activation of macropinocytosis. Methods: Using HeLa cells we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Results: Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell centre. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in macropinocytosis. Also, Rab34 induced repositioning of lysosomes does not affect transport of the mannose 6-phosphate receptor to endosomes. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34, or following RNA interference, failed to transport the temperature-sensitive Vesicular Stomatitis Virus G-protein fused to GFP (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous MHC class I (MHC) as a marker, an endoglycosidase H resistance assay showed that ER to medial Golgi traffic remains intact in knock-down cells indicating that Rab34 specifically functions in post-Golgi transport. Further, brefeldin A treatment revealed that Rab34 acts at the Golgi, not the trans-Golgi network. Conclusion: Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

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