scholarly journals A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

2020 ◽  
Author(s):  
Kaitlyn McGrath ◽  
Mykola Dergai ◽  
Shivani Agarwal ◽  
Daayun Chung ◽  
Damian B. van Rossum ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Conformational Change ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Structural Modeling ◽  
Snare Proteins ◽  
Conformational Switch ◽  
Membrane Bound ◽  
Open Membrane

ABSTRACTMembrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. The phosphorylation site we identified is highly conserved in evolution and is regulated by the Ca2+-dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel mechanism by which Ykt6 conformation and activity is regulated by Ca2+ signaling with implications in Parkinson’s Disease in which Ykt6 has been shown to play a role.

scholarly journals A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6

2021 ◽  
Vol 118 (12) ◽  
pp. e2016730118
Author(s):  
Kaitlyn McGrath ◽  
Shivani Agarwal ◽  
Marco Tonelli ◽  
Mykola Dergai ◽  
Anthony L. Gaeta ◽  
...  
Keyword(s):  
Conformational Change ◽  
Protein Interactions ◽  
Multidisciplinary Approach ◽  
Open Form ◽  
Conformational Switch ◽  
Protein Receptor ◽  
Evolutionarily Conserved ◽  
Sensitive Factor ◽  
Membrane Bound ◽  
Open Membrane

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson’s disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

scholarly journals Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells

2009 ◽  
Vol 20 (1) ◽  
pp. 338-347 ◽  
Author(s):  
Mark T. Breckenridge ◽  
Natalya G. Dulyaninova ◽  
Thomas T. Egelhoff
Keyword(s):  
Conformational Change ◽  
Mammalian Cells ◽  
Myosin Ii ◽  
Phosphorylation Site ◽  
Leading Edge ◽  
Live Cells ◽  
Nonmuscle Myosin ◽  
Related Disorder ◽  
Filament Assembly ◽  
Nonmuscle Myosin Ii

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (ΔIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Δtailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Δtailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.

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 Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

scholarly journals SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Nogueira ◽  
Patrik Erlmann ◽  
Julien Villeneuve ◽  
António JM Santos ◽  
Emma Martínez-Alonso ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Secretion ◽  
Family Members ◽  
Retrograde Transport ◽  
Cytoplasmic Domain ◽  
Snare Complex ◽  
Snare Proteins ◽  
General Protein

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.

scholarly journals Why are SNAREpins rod-shaped?

2021 ◽  
Author(s):  
Jin Zeng ◽  
Zachary McDargh ◽  
Dong An ◽  
Ben O'Shaughnessy
Keyword(s):  
Membrane Fusion ◽  
Strong Support ◽  
Force Production ◽  
Snare Proteins ◽  
Coarse Grained ◽  
Aspect Ratios ◽  
Extended Contact ◽  
Fusion Site ◽  
Simple Body ◽  
Core Components

SNARE proteins are the core components of the cellular machineries that fuse membranes for neurotransmitter or hormone release and other fundamental processes. Fusion is accomplished when SNARE proteins hosted by apposing membranes form SNARE complexes called SNAREpins, but the mechanism of fusion remains unclear. Computational simulations of SNARE-mediated membrane fusion are challenging due to the millisecond timescales of physiological membrane fusion. Here we used ultra-coarse-grained (UCG) simulations to investigate the minimal requirements for a molecular intracellular fusogen, and to elucidate the mechanisms of SNARE-mediated fusion. We find fusion by simple body forces that push vesicles together is highly inefficient. Inter-vesicle fusogens with different aspect ratios can fuse vesicles only if they are rodlike, of sufficient length to clear the fusogens from the fusion site by entropic forces. Simulations with rod-shaped SNAREpin-like fusogens fused 50-nm vesicles on ms timescales, driven by entropic forces along a reproducible fusion pathway. SNARE-SNARE and SNARE-membrane entropic forces cleared the fusion site and pressed the vesicles into an extended contact zone (ECZ), drove stalk nucleation at the high curvature ECZ boundary, and expanded the stalk into a long-lived hemifusion diaphragm in which a simple pore completed fusion. Our results provide strong support for the entropic hypothesis of SNARE-mediated membrane fusion, and implicate the rodlike structure of the SNAREpin complex as a necessity for entropic force production and fusion.

scholarly journals Relationships between the Activitiesin Vitroandin Vivoof Various Kinds of Ribozyme and Their Intracellular Localization in Mammalian Cells

2001 ◽  
Vol 276 (18) ◽  
pp. 15378-15385 ◽  
Author(s):  
Yoshio Kato ◽  
Tomoko Kuwabara ◽  
Masaki Warashina ◽  
Hirofumi Toda ◽  
Kazunari Taira
Keyword(s):  
Mammalian Cells ◽  
Intracellular Localization

Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation

2008 ◽  
Vol 412 (3) ◽  
pp. 563-577 ◽  
Author(s):  
Stine K. Knudsen ◽  
Allan Stensballe ◽  
Magnus Franzmann ◽  
Uffe B. Westergaard ◽  
Daniel E. Otzen
Keyword(s):  
Mammalian Cells ◽  
Bacterial Attachment ◽  
Passenger Domain ◽  
Glycosylation Sites ◽  
Structural And Functional Properties ◽  
Cellular Aggregation ◽  
Membrane Bound ◽  
Extracellular Environment ◽  
Resistant Structure ◽  
Different Levels

Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound β-domain and a passenger domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several passenger domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. In the present study we describe the effect of glycosylation on the extracellular passenger domain of the Escherichia coli autotransporter Ag43α, which induces frizzy colony morphology and cell settling. We identify 16 glycosylation sites and suggest two possible glycosylation motifs for serine and threonine residues. Glycosylation stabilizes against thermal and chemical denaturation and increases refolding kinetics. Unexpectedly, glycosylation also reduces the stabilizing effect of Ca2+ ions, removes the ability of Ca2+ to promote cell adhesion, reduces the ability of Ag43α-containing cells to form bacterial amyloid and increases the susceptibility of the resulting amyloid to proteolysis. In addition, our results indicate that Ag43α folds without a stable intermediate, unlike pertactin, indicating that autotransporters may arrive at the native state by a variety of different mechanisms despite a common overall structure. A small but significant fraction of Ag43α can survive intact in the periplasm if expressed without the β-domain, suggesting that it is able to adopt a protease-resistant structure prior to translocation across the membrane. The present study demonstrates that glycosylation may play significant roles in structural and functional properties of bacterial autotransporters at many different levels.

Delivering Ion Channels to Mammalian Cells by Membrane Fusion

2003 ◽  
pp. 275-296
Author(s):  
David C. Johns ◽  
Uta C. Hoppe ◽  
Eduardo Marbán ◽  
Brian O'Rourke
Keyword(s):  
Ion Channels ◽  
Membrane Fusion ◽  
Mammalian Cells
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