scholarly journals Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

1997 ◽  
Vol 139 (5) ◽  
pp. 1097-1108 ◽  
Author(s):  
Charles Barlowe
Keyword(s):  
Golgi Complex ◽  
Intact Cell ◽  
Vesicle Fusion ◽  
Intact Cells ◽  
Vesicle Budding ◽  
Vesicle Docking ◽  
Golgi Membranes ◽  
Golgi Transport ◽  
A Cell ◽  
A Subunit

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

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.

scholarly journals Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex.

1992 ◽  
Vol 118 (6) ◽  
pp. 1333-1345 ◽  
Author(s):  
I Corthésy-Theulaz ◽  
A Pauloin ◽  
S R Pfeffer
Keyword(s):  
Golgi Complex ◽  
Atp Hydrolysis ◽  
Intact Cell ◽  
Cytoplasmic Dynein ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Intact Cells ◽  
Organizing Center ◽  
Cell Cytosol ◽  
Stable Association

The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.

scholarly journals Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

1993 ◽  
Vol 4 (4) ◽  
pp. 413-424 ◽  
Author(s):  
L C Hendricks ◽  
M McCaffery ◽  
G E Palade ◽  
M G Farquhar
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Acinar Cells ◽  
Atp Depletion ◽  
Pancreatic Acinar Cells ◽  
Coat Proteins ◽  
Golgi Membranes ◽  
Golgi Transport ◽  
Transport Vesicles

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.

scholarly journals Golgi Enzymes Are Enriched in Perforated Zones of Golgi Cisternae but Are Depleted in COPI Vesicles

2004 ◽  
Vol 15 (10) ◽  
pp. 4710-4724 ◽  
Author(s):  
Hee-Seok Kweon ◽  
Galina V. Beznoussenko ◽  
Massimo Micaroni ◽  
Roman S. Polishchuk ◽  
Alvar Trucco ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Three Dimensional ◽  
Retrograde Transport ◽  
Cell Types ◽  
Free System ◽  
Progression Model ◽  
Golgi Transport ◽  
A Cell ◽  
Different Cell Types

In the most widely accepted version of the cisternal maturation/progression model of intra-Golgi transport, the polarity of the Golgi complex is maintained by retrograde transport of Golgi enzymes in COPI-coated vesicles. By analyzing enzyme localization in relation to the three-dimensional ultrastructure of the Golgi complex, we now observe that Golgi enzymes are depleted in COPI-coated buds and 50- to 60-nm COPI-dependent vesicles in a variety of different cell types. Instead, we find that Golgi enzymes are concentrated in the perforated zones of cisternal rims both in vivo and in a cell-free system. This lateral segregation of Golgi enzymes is detectable in some stacks during steady-state transport, but it was significantly prominent after blocking endoplasmic reticulum-to-Golgi transport. Delivery of transport carriers to the Golgi after the release of a transport block leads to a diminution in Golgi enzyme concentrations in perforated zones of cisternae. The exclusion of Golgi enzymes from COPI vesicles and their transport-dependent accumulation in perforated zones argues against the current vesicle-mediated version of the cisternal maturation/progression model.

scholarly journals Targeting of a Tropomyosin Isoform to Short Microfilaments Associated with the Golgi Complex

2004 ◽  
Vol 15 (1) ◽  
pp. 268-280 ◽  
Author(s):  
Justin M. Percival ◽  
Julie A. I. Hughes ◽  
Darren L. Brown ◽  
Galina Schevzov ◽  
Kirsten Heimann ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Actin Filaments ◽  
Brefeldin A ◽  
Cytochalasin D ◽  
Stress Fibers ◽  
G1 Phase ◽  
Vesicle Budding ◽  
Golgi Membranes ◽  
Internal Exon ◽  
Alternatively Spliced

A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as γ-Tm), but not from either the α- or β-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., ( 1999 ). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.

scholarly journals Alpha-SNAP but not gamma-SNAP is required for ER-Golgi transport after vesicle budding and the Rab1-requiring step but before the EGTA-sensitive step

1998 ◽  
Vol 111 (17) ◽  
pp. 2625-2633 ◽  
Author(s):  
F. Peter ◽  
S.H. Wong ◽  
V.N. Subramaniam ◽  
B.L. Tang ◽  
W. Hong
Keyword(s):  
Marked Contrast ◽  
In Vitro System ◽  
Vesicle Budding ◽  
Vesicle Docking ◽  
Golgi Transport ◽  
Site Of Action ◽  
Transport Assay ◽  
Attachment Proteins ◽  
Normal Transport

N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) have been implicated in diverse vesicular transport events; yet their exact role and site of action remain to be established. Using an established in vitro system, we show that antibodies against alpha-SNAP inhibit vesicle transport from the ER to the cis-Golgi and that recombinant alpha-SNAP enhances/stimulates the process. Cytosol immunodepleted of alpha-SNAP does not support normal transport unless supplemented with recombinant alpha-SNAP but not gamma-SNAP. In marked contrast, cytosol immunodepleted of gamma-SNAP supports ER-Golgi transport to the normal level. Neither antibodies against gamma-SNAP nor recombinant gamma-SNAP have any effect on ER-Golgi transport. These results clearly establish an essential role for alpha-SNAP but not gamma-SNAP in ER-Golgi transport. When the transport assay is performed with cytosol immunodepleted of alpha-SNAP, followed by incubation with cytosol immunodepleted of a COPII subunit, normal transport is achieved. In marked contrast, no transport is detected when the assay is first performed with cytosol depleted of the COPII subunit followed by alpha-SNAP-depleted cytosol, suggesting that alpha-SNAP is required after a step that requires COPII (the budding step). In combination with cytosol immunodepleted of Rab1, it is seen that alpha-SNAP is required after a Rab1-requiring step. It has been shown previously that EGTA blocks ER-Golgi transport at a step after vesicle docking but before fusion and we show here that alpha-SNAP acts before the step that is blocked by EGTA. Our results suggest that alpha-SNAP may be involved in the pre-docking or docking but not the fusion process.

scholarly journals Binding of the cytosolic p200 protein to Golgi membranes is regulated by heterotrimeric G proteins

1993 ◽  
Vol 106 (4) ◽  
pp. 1239-1248 ◽  
Author(s):  
J.B. de Almeida ◽  
J. Doherty ◽  
D.A. Ausiello ◽  
J.L. Stow
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Golgi Complex ◽  
Membrane Binding ◽  
Secretory Proteins ◽  
Coat Proteins ◽  
Heterotrimeric G Proteins ◽  
Intact Cells ◽  
Golgi Membranes

The formation of vesicles for protein trafficking requires the dynamic binding of cytosolic coat proteins onto Golgi membranes and this binding is regulated by a variety of GTPases, including heterotrimeric G proteins. We have previously shown the presence of the pertussis toxin-sensitive G alpha i-3 protein on Golgi membranes and demonstrated a functional role for G alpha i-3 in the trafficking of secretory proteins through the Golgi complex. We have also described a brefeldin A-sensitive phosphoprotein, p200, which is found in the cytoplasm and on Golgi membranes. The present study investigates the role of heterotrimeric G proteins in the regulation of p200 binding to Golgi membranes. An in vitro binding assay was used to measure the binding of cytosolic p200 to LLC-PK1 cell microsomal membranes and to purified rat liver Golgi membranes in the presence of specific activators of G proteins. The binding of p200 to Golgi membranes was compared to that of the coatomer protein beta-COP, for which G protein-dependent membrane binding has previously been established. Membrane binding of both p200 and beta-COP was induced maximally by activation of all G proteins in the presence of GTP gamma S. More selective activation of the heterotrimeric G proteins, with AlFn or mastoparan, also induced membrane binding of p200 and beta-COP. Pertussis toxin pretreatment of Golgi membranes, to selectively inactivate G alpha i-3, reduced the AlFn and mastoparan-induced binding of p200 to Golgi membranes, whereas no significant effect of pertussis toxin on beta-COP binding was found in this assay. The effect of pertussis toxin thus implicates G alpha i-3, as one component of a regulatory pathway, in the binding of cytosolic p200 to Golgi membranes. The effects of AlFn and pertussis toxin on p200 membrane binding were also shown in intact cells by immunofluorescence staining. AlFn treatment of cells induced translocation of p200 from the cytoplasm onto the Golgi complex, resulting in a conformational change in some Golgi membranes. The translocation of p200 was blocked by pretreatment of intact NRK cells with pertussis toxin. The data presented here support the conclusion that the binding of the p200 protein to Golgi membranes involves regulation by the pertussis toxin-sensitive heterotrimeric G proteins, specifically the G alpha i-3 protein.

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