scholarly journals Reconstitution of vesiculated Golgi membranes into stacks of cisternae: requirement of NSF in stack formation.

1995 ◽  
Vol 129 (3) ◽  
pp. 577-589 ◽  
Author(s):  
U Acharya ◽  
J M McCaffery ◽  
R Jacobs ◽  
V Malhotra
Keyword(s):  
High Speed ◽  
Protein Transport ◽  
Rat Kidney ◽  
Average Diameter ◽  
Cell Extract ◽  
Processing Enzymes ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Target Membrane

We have developed an in vitro system to study the biochemical events in the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae. The Golgi complex in intact normal rat kidney cells (NRK) is vesiculated by treatment with IQ. The cells are washed to remove the drug and then permeabilized by a rapid freeze-thaw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires ATP and a high speed supernatant from cell extract (cytosol), as revealed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and contain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to promote fusion of transport vesicles with the target membrane in the exocytic and endocytic pathways. Immunoelectron microscopy using Golgi specific anti-mannosidase II antibody reveals that VGMs undergo sequential changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.

scholarly journals Characteristics of endoplasmic reticulum-derived transport vesicles.

1994 ◽  
Vol 126 (5) ◽  
pp. 1133-1148 ◽  
Author(s):  
M F Rexach ◽  
M Latterich ◽  
R W Schekman
Keyword(s):  
Membrane Proteins ◽  
Protein Transport ◽  
De Novo ◽  
Microscopic Analysis ◽  
Equilibrium Density ◽  
Protein Markers ◽  
Electron Microscopic ◽  
Golgi Membranes ◽  
Transport Vesicles

We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.

scholarly journals The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.

1992 ◽  
Vol 118 (6) ◽  
pp. 1321-1332 ◽  
Author(s):  
B W Wattenberg ◽  
T J Raub ◽  
R R Hiebsch ◽  
P J Weidman
Keyword(s):  
Protein Transport ◽  
Vesicle Formation ◽  
Attachment Protein ◽  
Direct Role ◽  
Transport Vesicles ◽  
Sensitive Factor ◽  
Fusion Site ◽  
Transport Vesicle ◽  
Target Membrane ◽  
A Cell

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.

scholarly journals Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor.

1989 ◽  
Vol 108 (5) ◽  
pp. 1589-1596 ◽  
Author(s):  
P J Weidman ◽  
P Melançon ◽  
M R Block ◽  
J E Rothman
Keyword(s):  
Fusion Protein ◽  
Protein S ◽  
Membrane Receptor ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
A Cell ◽  
Free Transport ◽  
Affinity Interaction ◽  
Cytosolic Factor

An N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) has recently been purified on the basis of its ability to restore transport to NEM-inactivated Golgi membranes in a cell-free transport system. NSF is a peripheral membrane protein required for the fusion of transport vesicles. We now report the existence of two novel components that together bind NSF to Golgi membranes in a saturable manner. These components were detected by examining the requirements for reassociation of purified NSF with Golgi membranes in vitro. One component is an integral membrane receptor that is heat sensitive, but resistant to Na2CO3 extraction and to all proteases tested. The second component is a cytosolic factor that is sensitive to both proteases and heat. This soluble NSF attachment protein (SNAP) is largely resistant to NEM and is further distinguished from NSF by chromatography. SNAP appears to act stoichiometrically in promoting a high-affinity interaction between NSF and the membrane receptor. Because NSF promotes vesicle fusion, it seems likely that these two new factors that allow NSF to bind to the membrane are also part of the fusion machinery.

scholarly journals Identification of a 25-kD protein from yeast cytosol that operates in a prefusion step of vesicular transport between compartments of the Golgi.

1990 ◽  
Vol 110 (4) ◽  
pp. 947-954 ◽  
Author(s):  
B W Wattenberg ◽  
R R Hiebsch ◽  
L W LeCureux ◽  
M P White
Keyword(s):  
Monoclonal Antibodies ◽  
Golgi Apparatus ◽  
Active Component ◽  
Protein Transport ◽  
Transport Activity ◽  
Yeast Protein ◽  
Fusion Event ◽  
Transport Vesicles ◽  
Target Membrane ◽  
Cytosolic Factors

We have identified a 25-kD cytosolic yeast protein that mediates a late, prefusion step in transport of proteins between compartments of the Golgi apparatus. Activity was followed using the previously described cell free assay for protein transport between Golgi compartments as modified to detect late acting cytosolic factors (Wattenberg, B. W., and J. E. Rothman. 1986. J. Biol. Chem. 263:2208-2213). In the reaction mediated by this protein, transport vesicles that have become attached to the target membrane during a preincubation are processed in preparation for fusion. The ultimate fusion event does not require the addition of cytosolic proteins (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:525-536). Although isolated from yeast, this protein has activity when assayed with mammalian membranes. This protein has been enriched over 150-fold from yeast cytosol, albeit not to complete homogeneity. The identity of a 25-kD polypeptide as the active component was confirmed by raising monoclonal antibodies to it. These antibodies were found to specifically inhibit transport activity. Because this is a protein operating in prefusion, it has been abbreviated POP.

scholarly journals Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER.

1991 ◽  
Vol 114 (4) ◽  
pp. 663-670 ◽  
Author(s):  
C d'Enfert ◽  
L J Wuestehube ◽  
T Lila ◽  
R Schekman
Keyword(s):  
Membrane Protein ◽  
Protein Transport ◽  
Binding Protein ◽  
Integral Membrane Protein ◽  
Gtp Binding Protein ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Transport Vesicles

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.

scholarly journals Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN

1997 ◽  
Vol 138 (2) ◽  
pp. 291-306 ◽  
Author(s):  
Anne Müsch ◽  
David Cohen ◽  
Enrique Rodriguez-Boulan
Keyword(s):  
Mdck Cells ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Vesicle Release ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Nonmuscle Myosin Ii ◽  
Vesicular Carriers ◽  
Vesicular Stomatitis

The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113–1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II could be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibited the release of VSVG-containing vesicles from the TGN.

scholarly journals Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast

1994 ◽  
Vol 124 (4) ◽  
pp. 425-434 ◽  
Author(s):  
T Oka ◽  
A Nakano
Keyword(s):  
Protein Transport ◽  
Active Form ◽  
Integral Membrane Protein ◽  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Vesicles ◽  
In Vitro Reconstitution

The SAR1 gene product (Sar1p), a 21-kD GTPase, is a key component of the ER-to-Golgi transport in the budding yeast. We previously reported that the in vitro reconstitution of protein transport from the ER to the Golgi was dependent on Sar1p and Sec12p (Oka, T., S. Nishikawa, and A. Nakano. 1991. J. Cell Biol. 114:671-679). Sec12p is an integral membrane protein in the ER and is essential for the Sar1 function. In this paper, we show that Sar1p can remedy the temperature-sensitive defect of the sec12 mutant membranes, which is in the formation of ER-to-Golgi transport vesicles. The addition of Sar1p promotes vesicle formation from the ER irrespective of the GTP- or GTP gamma S-bound form, indicating that the active form of Sar1p but not the hydrolysis of GTP is required for this process. The inhibition of GTP hydrolysis blocks transport of vesicles to the Golgi and thus causes their accumulation. The accumulating vesicles, which carry Sar1p on them, can be separated from other membranes, and, after an appropriate wash that removes Sar1p, are capable of delivering the content to the Golgi when added back to fresh membranes. Thus we have established a new method for isolation of functional intermediate vesicles in the ER-to-Golgi transport. The sec23 mutant is defective in activation of Sar1 GTPase (Yoshihisa, T., C. Barlowe, and R. Schekman. 1993. Science (Wash. DC). 259:1466-1468). The membranes and cytosol from the sec23 mutant show only a partial defect in vesicle formation and this defect is also suppressed by the increase of Sar1p. Again GTP hydrolysis is not needed for the suppression of the defect in vesicle formation. Based on these results, we propose a model in which Sar1p in the GTP-bound form is required for the formation of transport vesicles from the ER and the GTP hydrolysis by Sar1p is essential for entering the next step of vesicular transport to the Golgi apparatus.

Fabrication of random and aligned-oriented cellulose acetate nanofibers containing betamethasone sodium phosphate: structural and cell biocompatibility evaluations

2017 ◽  
Vol 37 (9) ◽  
pp. 911-920 ◽  
Author(s):  
Saba Saifoori ◽  
Mahshid Fallah-Darrehchi ◽  
Payam Zahedi ◽  
Abdolmajid Bayandori Moghaddam
Keyword(s):  
Cellulose Acetate ◽  
High Speed ◽  
Sodium Phosphate ◽  
Phosphate Buffer Solution ◽  
Average Diameter ◽  
Burst Release ◽  
Buffer Solution ◽  
Hematoxylin And Eosin ◽  
Betamethasone Sodium Phosphate

Abstract: The objective of this work was to prepare electrospun cellulose acetate (CA) nanofibers containing betamethasone sodium phosphate (BSP). Two different morphologies including random and aligned orientations were rationally designed to improve the performance of samples in in vitro experiments. By comparing the CA nanofibrous samples with randomly and aligned-oriented morphologies, the scanning electron microscopy images showed that the neat aligned-oriented nanofibers with an average diameter of 180±15 nm could be obtained using a high-speed rotating collector. Subsequently, the tensile test confirmed that the aligned CA nanofibers had higher mechanical properties than that of the randomly oriented ones. Moreover, the BSP release profile obtained by UV-vis spectrophotometry depicted that the aligned samples had an initial burst release of BSP followed by a slow penetration of the drug with a gentle slope during 72 h. Furthermore, the ultimate amounts of BSP released from the random and aligned CA nanofibers into the phosphate buffer solution were 63% and 53%, respectively. Finally, human adipose-derived mesenchymal stem cells were seeded on both aligned and random electrospun CA nanofibrous samples containing BSP. The thiazolyl blue and hematoxylin and eosin staining results showed that the BSP-loaded nanofibers with the aligned morphology provided the most suitable environment for the cells’ growth, viability, and proliferation.

scholarly journals Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

2004 ◽  
Vol 167 (6) ◽  
pp. 997-1003 ◽  
Author(s):  
Dalu Xu ◽  
Jesse C. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Secretory Pathway ◽  
Golgi Stack ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Intermediate Compartment ◽  
Copii Vesicles

What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

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