scholarly journals Phosphorylation of the Vesicle-Tethering Protein P115 by a Casein Kinase II–Like Enzyme Is Required for Golgi Reassembly from Isolated Mitotic Fragments

2000 ◽  
Vol 150 (3) ◽  
pp. 475-488 ◽  
Author(s):  
A. Barbara Dirac-Svejstrup ◽  
James Shorter ◽  
M. Gerard Waters ◽  
Graham Warren
Keyword(s):  
Coiled Coil ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Free System ◽  
Vesicle Docking ◽  
Vesicle Tethering ◽  
Receptor Complexes ◽  
Transport Vesicles ◽  
A Cell ◽  
Copi Vesicle

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH2-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide–sensitive fusion protein)–catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of t-SNARE (trans–soluble NSF attachment protein [SNAP] receptor) complexes.

scholarly journals Selective degradation of insulin within rat liver endosomes.

1990 ◽  
Vol 110 (1) ◽  
pp. 35-42 ◽  
Author(s):  
J J Doherty ◽  
D G Kay ◽  
W H Lai ◽  
B I Posner ◽  
J J Bergeron
Keyword(s):  
Significant Inhibition ◽  
Cell Fraction ◽  
Insulin Degradation ◽  
Free System ◽  
Cell Free System ◽  
Selective Degradation ◽  
Receptor Complexes ◽  
Specific Protease ◽  
A Cell ◽  
Rapid Generation

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.

scholarly journals Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

1997 ◽  
Vol 17 (2) ◽  
pp. 594-603 ◽  
Author(s):  
S C Nair ◽  
R A Rimerman ◽  
E J Toran ◽  
S Chen ◽  
V Prapapanich ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Steroid Receptors ◽  
Free System ◽  
Isomerase Activity ◽  
Receptor Complexes ◽  
Wide Range ◽  
A Cell ◽  
Cyclophilin 40 ◽  
Peptidylprolyl Isomerase

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.

scholarly journals Reconstitution and Analysis of Soluble Inhibin and Activin Receptor Complexes in a Cell-free System

2004 ◽  
Vol 279 (51) ◽  
pp. 53126-53135 ◽  
Author(s):  
Elisabetta del Re ◽  
Yisrael Sidis ◽  
David A. Fabrizio ◽  
Herbert Y. Lin ◽  
Alan Schneyer
Keyword(s):  
Free System ◽  
Activin Receptor ◽  
Cell Free System ◽  
Receptor Complexes ◽  
A Cell

scholarly journals A Role for the Vesicle Tethering Protein, P115, in the Post-Mitotic Stacking of Reassembling Golgi Cisternae in a Cell-Free System

1999 ◽  
Vol 146 (1) ◽  
pp. 57-70 ◽  
Author(s):  
James Shorter ◽  
Graham Warren
Keyword(s):  
Temporal Analysis ◽  
Heterogeneous Population ◽  
Initial Alignment ◽  
Attachment Protein ◽  
Free System ◽  
Cell Free System ◽  
Vesicle Tethering ◽  
Protein Receptors ◽  
A Cell ◽  
Membrane Bound

During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)–sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

scholarly journals COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system.

1994 ◽  
Vol 125 (2) ◽  
pp. 269-282 ◽  
Author(s):  
T Misteli ◽  
G Warren
Keyword(s):  
Strong Support ◽  
Serial Sectioning ◽  
Animal Cells ◽  
Cdc2 Kinase ◽  
Cross Sectional ◽  
Free System ◽  
Transport Vesicles ◽  
A Cell ◽  
Gamma S ◽  
Total Membrane

Rat liver Golgi stacks fragmented when incubated with mitotic but not interphase cytosol in a process dependent on time, temperature, energy (added in the form of ATP) and cdc2 kinase. The cross-sectional length of Golgi stacks fell in the presence of mitotic cytosol by approximately 50% over 30 min without a corresponding decrease in the number of cisternae in the stack. The loss of membrane from stacked and single cisternae occurred with a half-time of approximately 20 min, and was matched by the appearance of both small (50-100 nm in diameter) and large (100-200 nm in diameter) vesicular profiles. Small vesicular profiles constituted more than 50% of the total membrane after 60 min of incubation and they were shown to be vesicles or very short tubules by serial sectioning. In the presence of GTP gamma S all of the small vesicles were COP-coated and both the extent and the rate at which they formed were sufficient to account for the production of small vesicles during mitotic incubation. The involvement of the COP-mediated budding mechanism was confirmed by immunodepletion of one of the subunits of COP coats (the coatomer) from mitotic cytosol. Vesicles were no longer formed but highly fenestrated networks appeared, an effect reversed by the readdition of purified coatomer. Together these experiments provide strong support for our hypothesis that the observed vesiculation of the Golgi apparatus during mitosis in animal cells is caused by continued budding of COP-coated transport vesicles but an inhibition of their fusion with their target membranes.

scholarly journals GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering

2011 ◽  
Vol 194 (5) ◽  
pp. 779-787 ◽  
Author(s):  
Frank C. Brown ◽  
Carmel H. Schindelhaim ◽  
Suzanne R. Pfeffer
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
Vesicle Tethering ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Transport Vesicle ◽  
Golgi Structure ◽  
Clathrin Adaptor ◽  
Golgi Network

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.

scholarly journals Protein flexibility is required for vesicle tethering at the Golgi

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Pak-yan Patricia Cheung ◽  
Charles Limouse ◽  
Hideo Mabuchi ◽  
Suzanne R Pfeffer
Keyword(s):  
Coiled Coil ◽  
Rab Gtpases ◽  
Force Microscopy ◽  
Vesicle Tethering ◽  
Proximity Ligation ◽  
Atomic Force ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Specific Sequences

The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering.

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