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 Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

scholarly journals Regulation of heat shock protein synthesis by quercetin in human erythroleukaemia cells

1994 ◽  
Vol 300 (1) ◽  
pp. 201-209 ◽  
Author(s):  
G Elia ◽  
M G Santoro
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Elevated Temperatures ◽  
K562 Cells ◽  
Hsp70 Expression ◽  
Transcriptional Level ◽  
Hsp70 Mrna ◽  
Shock Proteins ◽  
Hsp70 Synthesis

Synthesis of heat-shock proteins (HSPs) is universally induced in eukaryotic and prokaryotic cells by exposure to elevated temperatures or to other types of environmental stress. In mammalian cells, HSPs belonging to the 70 kDa family (HSP70) have a regulatory role in several cellular processes, and have been shown to be involved in the control of cell proliferation and differentiation. Although many types of HSP70 inducers have been identified, only a few compounds, all belonging to the flavonoid group, have been shown to inhibit HSP70 induction. Because inhibitors of HSP70 synthesis could be an important tool with which to study the function of this protein, we have investigated the effect of quercetin, a flavonoid with antiproliferative activity which is widely distributed in nature, on HSP70 synthesis in human K562 erythroleukaemia cells after treatment with severe or mild heat shock and with other inducers. Quercetin was found to affect HSP70 synthesis at more than one level, depending on the conditions used. Indeed, after severe heat shock (45 degrees C for 20 min) treatment with quercetin, at non-toxic concentrations, was found to inhibit HSP70 synthesis for a period of 3-4 h. This block appeared to be exerted at the post-transcriptional level and to be cell-mediated, as the addition of quercetin during translation of HSP70 mRNA in vitro had no effect. After prolonged (90 min) exposure at 43 degrees C, however, quercetin was found to inhibit also HSP70 mRNA transcription. Pretreatment of K562 cells with quercetin had no effect on HSP70 expression, and quercetin needed to be present during induction to be effective. Under all conditions tested, the quercetin-induced block of HSP70 synthesis was found to be transient and, after an initial delay, synthesis of HSP70 reached the control rate and continued at the same level for several hours after the time at which HSP70 synthesis had been turned off in control cells. Finally, inhibition of HSP70 synthesis by quercetin appeared to be dependent on the temperature used and on the type of stressor.

Expression of a Drosophila heat shock protein in mammalian cells: transient association with nucleoli after heat shock

1984 ◽  
Vol 307 (1132) ◽  
pp. 301-307 ◽  
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Mammalian Cells ◽  
Actinomycin D ◽  
Future Studies ◽  
Cos Cells ◽  
Single Protein ◽  
Cellular Components ◽  
Multiple Interactions

We transformed mouse L cells with a cloned Drosophila hsp70 gene and obtained cells with either heat-inducible or constitutively expressed copies of the gene. The distribution of hsp70 in these cells was examined by indirect immunofluorescence with monoclonal antibodies specific to the Drosophila protein. In constitutive cells, hsp70 was present in both cytoplasm and nucleus. After heat shock, the nuclear hsp70 was transiently concentrated in nucleoli, from which it had previously been excluded; the cytoplasmic hsp70 moved to a perinuclear location, a result consistent with it being associated with intermediate filaments of the cytoskeleton. The nucleolar migration took several hours and was partly inhibited by actinomycin D, but was independent of protein synthesis; it may reflect binding to newly-synthesized rRNA. Drosophila hsp70 was also expressed from replicating plasmids in monkey COS cells, and was found to be concentrated in nuclei even at low temperature. Migration to nucleoli occurred after heat shock. These results indicate that a single protein can have multiple interactions with cellular components, and form the basis for future studies of these interactions by in vitro mutagenesis and expression of the hsp70 gene.

scholarly journals Reconstitution in vitro of a membrane-fusion event involved in constitutive exocytosis. A role for cytosolic proteins and a GTP-binding protein, but not for Ca2+

1992 ◽  
Vol 285 (2) ◽  
pp. 383-385 ◽  
Author(s):  
J M Edwardson ◽  
P U Daniels-Holgate
Keyword(s):  
Membrane Fusion ◽  
Binding Protein ◽  
Fusion Event ◽  
Gtp Binding Protein ◽  
In Vitro System ◽  
Cytosolic Proteins ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Transport Vesicles

The fusion of post-Golgi transport vesicles with the plasma membrane is perhaps the least well understood step in the network of intracellular membrane traffic. We have used an ‘in vitro’ system to study this membrane-fusion event. We show here that fusion requires the presence of cytosolic proteins, but not Ca2+, and is inhibited by the non-hydrolysable GTP analogue guanosine 5′-[gamma-thio]triphosphate, which indicates the involvement of a GTP-binding protein.

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.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

2000 ◽  
Vol 113 (11) ◽  
pp. 1993-2002 ◽  
Author(s):  
H. Cao ◽  
H.M. Thompson ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Living Cells ◽  
Dominant Negative ◽  
Coated Vesicle ◽  
Large Numbers ◽  
Golgi Structure ◽  
And Function ◽  
Golgi Network ◽  
Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

scholarly journals Human STAGA Complex Is a Chromatin-Acetylating Transcription Coactivator That Interacts with Pre-mRNA Splicing and DNA Damage-Binding Factors In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 6782-6795 ◽  
Author(s):  
Ernest Martinez ◽  
Vikas B. Palhan ◽  
Agneta Tjernberg ◽  
Elena S. Lymar ◽  
Armin M. Gamper ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Binding Protein ◽  
Chromatin Modification ◽  
Hereditary Disease ◽  
Coupled Processes ◽  
Transcription Activators ◽  
Sequence Relationships

ABSTRACT GCN5 is a histone acetyltransferase (HAT) originally identified inSaccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAFII-containing complex], and STAGA [SPT3-TAFII31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.

Exercising mammals synthesize stress proteins

1990 ◽  
Vol 258 (4) ◽  
pp. C723-C729 ◽  
Author(s):  
M. Locke ◽  
E. G. Noble ◽  
B. G. Atkinson
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Sprague Dawley ◽  
Spleen Cells ◽  
Sprague Dawley Rats ◽  
Electrophoretic Separations ◽  
Shock Proteins ◽  
Sufficient Stimulus

Spleen cells, peripheral lymphocytes, and soleus muscles were removed from male Sprague-Dawley rats that had been run on a treadmill (24 m/min) for either 20, 40, or 60 min or to exhaustion (86 +/- 41 min) and were labeled in vitro with [35S]methionine at 37 degrees C. Similar tissues from nonrunning control rats were labeled in vitro at either 37 or 43 degrees C (heat shock). Fluorographic analyses of one- and two-dimensional polyacrylamide gel electrophoretic separations of the proteins from cells and tissues of exercised rats demonstrate the new or enhanced synthesis of proteins of approximately 65, 72, 90, and 100 kDa. Although synthesis of these proteins is low or not detectable in tissues from control rats labeled at 37 degrees C, they are prominent products of similar tissues labeled under heat-shock conditions (43 degrees C) and, in fact, correspond in Mr and pI with the so-called heat-shock proteins. These results suggest that exercise is a sufficient stimulus to induce or enhance the synthesis of heat shock and/or stress proteins in mammalian cells and tissues.

scholarly journals Cell cycle regulation of VCIP135 deubiquitinase activity and function in p97/p47-mediated Golgi reassembly

2015 ◽  
Vol 26 (12) ◽  
pp. 2242-2251 ◽  
Author(s):  
Xiaoyan Zhang ◽  
Yanzhuang Wang
Keyword(s):  
Cell Cycle ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Membrane Dynamics ◽  
Deubiquitinating Enzyme ◽  
Golgi Membrane ◽  
Interacting Protein ◽  
Daughter Cells ◽  
And Function ◽  
Cycle Regulation

In mammalian cells, the inheritance of the Golgi apparatus into the daughter cells during each cycle of cell division is mediated by a disassembly and reassembly process, and this process is precisely controlled by phosphorylation and ubiquitination. VCIP135 (valosin-containing protein p97/p47 complex–interacting protein, p135), a deubiquitinating enzyme required for p97/p47-mediated postmitotic Golgi membrane fusion, is phosphorylated at multiple sites during mitosis. However, whether phosphorylation directly regulates VCIP135 deubiquitinase activity and Golgi membrane fusion in the cell cycle remains unknown. We show that, in early mitosis, phosphorylation of VCIP135 by Cdk1 at a single residue, S130, is sufficient to inactivate the enzyme and inhibit p97/p47-mediated Golgi membrane fusion. At the end of mitosis, VCIP135 S130 is dephosphorylated, which is accompanied by the recovery of its deubiquitinase activity and Golgi reassembly. Our results demonstrate that phosphorylation and ubiquitination are coordinated via VCIP135 to control Golgi membrane dynamics in the cell cycle.

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