scholarly journals Sequential SNARE disassembly and GATE-16–GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion

2002 ◽  
Vol 157 (7) ◽  
pp. 1161-1173 ◽  
Author(s):  
Joyce M.M. Müller ◽  
James Shorter ◽  
Richard Newman ◽  
Katrin Deinhardt ◽  
Yuval Sagiv ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Atp Hydrolysis ◽  
Golgi Membrane ◽  
Complex Assembly ◽  
Evolutionarily Conserved ◽  
Golgi Fragmentation ◽  
Fusion Analysis

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/α-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.

scholarly journals DjA1 maintains Golgi integrity via interaction with GRASP65

2019 ◽  
Vol 30 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Jie Li ◽  
Danming Tang ◽  
Stephen C. Ireland ◽  
Yanzhuang Wang
Keyword(s):  
Heat Shock ◽  
Membrane Fusion ◽  
Structure Formation ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Golgi Membrane ◽  
Biochemical Methods ◽  
Golgi Fragmentation ◽  
Golgi Structure

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

scholarly journals Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

2016 ◽  
Vol 27 (1) ◽  
pp. 137-152 ◽  
Author(s):  
Danming Tang ◽  
Xiaoyan Zhang ◽  
Shijiao Huang ◽  
Hebao Yuan ◽  
Jie Li ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Elongation Factor ◽  
Golgi Membrane ◽  
Golgi Membranes ◽  
Golgi Fragmentation ◽  
Golgi Ribbon

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.

scholarly journals Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Sung-Eun Nam ◽  
Yiu Wing Sunny Cheung ◽  
Thanh Ngoc Nguyen ◽  
Michael Gong ◽  
Samuel Chan ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Late Endosome ◽  
Multisystem Disorder ◽  
Cytoplasmic Material ◽  
Disease Mutations ◽  
Evolutionarily Conserved ◽  
Overall Stability ◽  
Transport Vesicle ◽  
Molecular Effects

AbstractPivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.

An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion

10.1038/14025 ◽  
1999 ◽  
Vol 1 (6) ◽  
pp. 335-340 ◽  
Author(s):  
Joyce M. M. Müller ◽  
Catherine Rabouille ◽  
Richard Newman ◽  
James Shorter ◽  
Paul Freemont ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Golgi Membrane

scholarly journals Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

2003 ◽  
Vol 185 (15) ◽  
pp. 4442-4449 ◽  
Author(s):  
Gregory M. Cook ◽  
Stefanie Keis ◽  
Hugh W. Morgan ◽  
Christoph von Ballmoos ◽  
Ulrich Matthey ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Atp Synthesis ◽  
Intrinsic Property ◽  
Protein Sequencing ◽  
Hydrolysis Activity

ABSTRACT We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ε and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

scholarly journals Hemophagocytic lymphohistiocytosis caused by dominant-negative mutations in STXBP2 that inhibit SNARE-mediated membrane fusion

Blood ◽  
2015 ◽  
Vol 125 (10) ◽  
pp. 1566-1577 ◽  
Author(s):  
Waldo A. Spessott ◽  
Maria L. Sanmillan ◽  
Margaret E. McCormick ◽  
Nishant Patel ◽  
Joyce Villanueva ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Dominant Negative ◽  
Snare Complex ◽  
Complex Assembly ◽  
Mutant Proteins ◽  
Key Points ◽  
Lymphocyte Cytotoxicity ◽  
Dominant Negative Mutations

Key Points Monoallelic STXBP2 mutations affecting codon 65 impair lymphocyte cytotoxicity and contribute to hemophagocytic lymphohistiocytosis. Munc18-2R65Q/W mutant proteins function in a dominant-negative manner to impair membrane fusion and arrest SNARE-complex assembly.

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