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 Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.

1993 ◽  
Vol 120 (4) ◽  
pp. 865-875 ◽  
Author(s):  
N K Pryer ◽  
N R Salama ◽  
R Schekman ◽  
C A Kaiser
Keyword(s):  
Gel Filtration ◽  
Beta Subunits ◽  
Vesicle Formation ◽  
Multicopy Plasmid ◽  
Formation Reaction ◽  
Internal Repeat ◽  
Transport Vesicles ◽  
Vesicle Biogenesis ◽  
A Cell

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.

Relationship between endosomes and lysosomes

2001 ◽  
Vol 29 (4) ◽  
pp. 476-480 ◽  
Author(s):  
J. P. Luzio ◽  
B. M. Mullock ◽  
P. R. Pryor ◽  
M. R. Lindsay ◽  
D. E. James ◽  
...  
Keyword(s):  
Acid Hydrolases ◽  
Attachment Protein ◽  
Intact Cells ◽  
Vesicular Traffic ◽  
Vacuole Fusion ◽  
Late Endosomes ◽  
Sensitive Factor ◽  
A Cell ◽  
Hydrolysis Of ◽  
Direct Fusion

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a ‘cell stomach’, acting as a major site of hydrolysis of endocytosed macromolecules.

scholarly journals Formation and Turnover of NSF- and SNAP-containing “Fusion” Complexes Occur on Undocked, Clathrin-coated Vesicle–derived Membranes

1998 ◽  
Vol 9 (7) ◽  
pp. 1633-1647 ◽  
Author(s):  
Eileithyia Swanton ◽  
John Sheehan ◽  
Naomi Bishop ◽  
Stephen High ◽  
Philip Woodman
Keyword(s):  
Vesicular Transport ◽  
Snare Complex ◽  
Coated Vesicle ◽  
Transport Pathways ◽  
Vesicle Docking ◽  
Transport Vesicles ◽  
Transport Vesicle ◽  
Target Membrane ◽  
Vesicle Cycle

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle–derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

scholarly journals Cvt9/Gsa9 Functions in Sequestering Selective Cytosolic Cargo Destined for the Vacuole

2001 ◽  
Vol 153 (2) ◽  
pp. 381-396 ◽  
Author(s):  
John Kim ◽  
Yoshiaki Kamada ◽  
Per E. Stromhaug ◽  
Ju Guan ◽  
Ann Hefner-Gravink ◽  
...  
Keyword(s):  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Sensitive Mutant ◽  
Direct Role ◽  
Vacuole Membrane ◽  
Cytoplasmic Material ◽  
Transport Vesicle ◽  
Membrane Compartment ◽  
Selective Delivery

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection. We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

scholarly journals Biogenesis of γ-secretase early in the secretory pathway

2007 ◽  
Vol 179 (5) ◽  
pp. 951-963 ◽  
Author(s):  
Jinoh Kim ◽  
Bertrand Kleizen ◽  
Regina Choy ◽  
Gopal Thinakaran ◽  
Sangram S. Sisodia ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Evidence ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Transport Vesicles ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
Transient Intermediate ◽  
Important Enzyme

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.

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 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 Interaction of SNAREs with ArfGAPs Precedes Recruitment of Sec18p/NSF

2007 ◽  
Vol 18 (8) ◽  
pp. 2852-2863 ◽  
Author(s):  
Christina Schindler ◽  
Anne Spang
Keyword(s):  
Conformational Change ◽  
Atp Hydrolysis ◽  
Small Gtpase ◽  
Snare Complex ◽  
Coat Proteins ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Target Membrane

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of α-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the “activated” SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.

scholarly journals AtVPS45 Complex Formation at thetrans-Golgi Network

2000 ◽  
Vol 11 (7) ◽  
pp. 2251-2265 ◽  
Author(s):  
Diane C. Bassham ◽  
Anton A. Sanderfoot ◽  
Valentina Kovaleva ◽  
Haiyan Zheng ◽  
Natasha V. Raikhel
Keyword(s):  
Secretory Pathway ◽  
Gene Families ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Attachment Protein ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Target Membrane ◽  
Functional Complexity ◽  
Golgi Network

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide–sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.

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