Stacking Protein Research Articles

Self-healing properties of flaws in nanoscale materials: Effects of soft and hard molecular dynamics simulations and boundaries studied using a continuum mechanical model

Soft and hard boundary effects on the flaw in self-healing capabilities of the nanoscale copper clusters and biomaterials have been studied using molecular dynamics simulations as well as the theoretical analyses. When the copper nanocluster size decreases to a compatible magnitude to its flaw, different boundary conditions change the flaw self-healing capability and lead to different dislocation generation and atom rearrangement after the copper nanocluster is healed. The theoretical predictions for the copper nanocluster are in good agreement with the molecular dynamics simulations. Further theoretical investigations demonstrate that the mineral layer in biomaterials possess a high flaw self-healing capability because of the nanometer scale and natural soft boundaries caused by the stacking protein and aragonite layered structures.

The BTB-kelch Protein LZTR-1 Is a Novel Golgi Protein That Is Degraded upon Induction of Apoptosis

Members of the BTB-kelch superfamily play important roles during fundamental cellular processes, such as the regulation of cell morphology, migration, and gene expression. The BTB-kelch protein LZTR-1 is deleted in the majority of DiGeorge syndrome patients and is believed to act as a transcriptional regulator. However, functional and expression profiling studies of LZTR-1 have not been performed thus far. Therefore, we examined the subcellular localization and function of LZTR-1 to gain insights into its biological role. Analysis of the primary structure of the protein revealed six N-terminal kelch motifs and two BTB/POZ domains at the C terminus within LZTR-1. Confocal analysis of the subcellular distribution of LZTR-1 using the Golgi markers GM130, Golgin-97, and TGN46 identified a localization of LZTR-1 exclusively on the cytoplasmic surface of the Golgi network that is mediated by its second BTB/POZ domain. In contrast to most other BTB-kelch proteins, LZTR-1 did not co-localize with actin. Treatment with brefeldin A did not lead to redistribution of LZTR-1 to the endoplasmic reticulum but caused its relocalization in dispersed, punctuated structures that were also positive for GM130. These data demonstrate that LZTR-1 is a Golgi matrix-associated protein. Upon induction of apoptosis, LZTR-1 was phosphorylated on tyrosine residues and subsequently degraded; that could be rescued partially by the addition of the caspase inhibitor Z-VAD-fmk and the proteasome inhibitors lactacystin and MG132. Taken together, our experiments identify LZTR-1 as the first BTB-kelch protein that exclusively localizes to the Golgi network, and the binding of LZTR-1 to the Golgi complex is mediated by its second BTB/POZ domain.

Convergence of Cell Cycle Regulation and Growth Factor Signals on GRASP65

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

05/01014 Goal-oriented flexible sensing for higher diagnostic performance of nuclear plant instrumentation

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation.

Mapping the Functional Domains of the Golgi Stacking Factor GRASP65

The Golgi reassembly stacking protein (GRASP) family has been implicated in the stacking of Golgi cisternae and the regulation of Golgi disassembly/reassembly during mitosis in mammalian cells. GRASP65 is a dimer that can directly link adjacent surfaces through trans-oligomerization in a mitotically regulated manner. Here we show that the N-terminal GRASP domain (amino acids 1-201) is both necessary and sufficient for dimerization and trans-oligomerization but is not mitotically regulated. The C-terminal serine/proline-rich domain (amino acids 202-446) cannot dimerize nor can it link adjacent surfaces. It does, however, confer mitotic regulation on the GRASP domain through multiple sites phosphorylated by the mitotic kinases, cdc2/B1, and the polo-like kinase. Transient expression corroborated these results by showing that the GRASP domain alone inhibited mitotic fragmentation of the Golgi apparatus.

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis.

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.

GRASP65 gets the chop

Early in apoptosis, caspase 3 chops up the Golgi stacking protein GRASP65 to fragment the Golgi, according to results presented by Lane et al. on page 495. The fragmentation may cut the Golgi into manageable pieces, and allow dispersion of the pieces so that they can be incorporated into apoptotic bodies near the disintegrating plasma membrane. Figure Apoptotic cells (red) lack GRASP65 (green). Lane et al. map the relevant cleavage sites on GRASP65 to the COOH terminus of the protein. A GRASP65 protein with these sites mutated is no longer cleaved, and the mutant protein preserves ∼1/4 of the Golgi stacking after apoptosis induction. Given that GRASP65 is located only in the cis Golgi, it might be expected to protect at most half of the Golgi from a loss of stacking. Lane et al. suspect that other caspase substrates control degradation of the rest of the Golgi, although they show that a number of other known Golgi stacking proteins are not cleaved during apoptosis.The results with mutant GRASP65 underscore the in vivo importance of GRASP65 in construction of the Golgi, which was previously only hinted at in in vitro assays. This function may be performed by the cleavable COOH terminus of the protein, whereas the vesicle tethering function of GRASP65 relies on the NH2 terminus. The mechanisms by which the COOH terminus carries out its job, including any protein partners, remain unknown. ▪

Polo-like kinase is required for the fragmentation of pericentriolar Golgi stacks during mitosis.

The pericentriolar stacks of Golgi cisternae undergo extensive reorganization during mitosis in mammalian cells. GM130 and GRASP65 (Golgi reassembly stacking protein of 65 kDa) are Golgi-associated proteins that are targets of mitotic kinases, and they have also been implicated in the reorganization of the Golgi structure during cell division. Previous studies have reported that mitogen-activated protein kinase kinase-1 (MEK1) and Cdc2 protein kinases are involved in these dynamic changes in the Golgi structure. More recently, the mitotic polo-like kinase (Plk) has been shown to interact with and phosphorylate GRASP65. Here, we provide evidence that Plk is involved in the mitosis-specific fragmentation of the Golgi apparatus. The addition of kinase-defective Plk or immunodepletion of Plk disrupts the fragmentation process. Furthermore, Golgi fragmentation is inhibited by the addition of either full-length or truncated GRASP65. These findings suggest that phosphorylation of GRASP65 by Plk may be a critical event in the reorganization of the Golgi structure during mitosis.

Mitotic phosphorylation of Golgi reassembly stacking protein 55 by mitogen-activated protein kinase ERK2.

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.

Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2

Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein. GRASP65 appears to function in the postmitotic reassembly of Golgi stacks. In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also phosphorylate GRASP65. In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function. Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65. These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division.

Matrix proteins can generate the higher order architecture of the Golgi apparatus.

The Golgi apparatus in animal cells comprises a reticulum of linked stacks in the pericentriolar and often in the juxtanuclear regions of the cell. The unique architecture of this organelle is thought to depend on the cytoskeleton and cytoplasmic matrix proteins--the best characterized being the golgin family of fibrous, coiled-coil proteins and the GRASP family of stacking proteins. Here we show that these matrix proteins can be separated from oligosaccharide-modifying enzymes in the Golgi stack without affecting their ability to form a ribbon-like reticulum in the correct location near to the nucleus. Our data suggest that the Golgi is a structural scaffold that can exist independently of, but is normally populated by, the enzyme-containing membranes that modify transiting cargo. This new concept of the Golgi further indicates that the Golgi may be an autonomous organelle rather than one that is in simple dynamic equilibrium with the endoplasmic reticulum.

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.

A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

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.

GRASP65, a Protein Involved in the Stacking of Golgi Cisternae

NEM prevents mitotic reassembly of Golgi cisternae into stacked structures. The major target of NEM is a 65 kDa protein conserved from yeast to mammals. Antibodies to this protein and a recombinant form of it block cisternal stacking in a cell-free system, justifying its designation as a Golgi ReAssembly Stacking Protein (GRASP65). One of the two minor targets of NEM is GM130, previously implicated in the docking of transport vesicles and mitotic fragmentation of the Golgi stack. GRASP65 is complexed with GM130 and is tightly bound to Golgi membranes, even under mitotic conditions when both are heavily phosphorylated. These results link vesicle docking, stacking of Golgi cisternae, and the disruption of both of these interactions during mitosis.

Polyacrylamide gel electrophoresis with optical scanning, using multiphasic buffer systems: The stack

The zone of stacked protein in polyacrylamide gel electrophoresis (PAGE) generated by a multiphasic (discontinuous) buffer system was analyzed by continuous optical scanning during electrophoresis. In agreement with theoretical prediction, zone width was found to be constant with time; band area was linearly related to protein load; band dispersion, measured as the standard deviation (σ) of the concentration profile, was independent of current density between 1 and 8 mA/cm 2; and σ decreased with increasing ionic strength of the gel buffer. The protein concentration within the stack ranged from 13 to 34 mg/ml for β-lactoglobulin B of 36,000 mw. In lieu of the predicted uniform distribution (“square-wave” function) for peak shape, the observed values of kurtosis and skewness indicated a slightly flattened Gaussian profile of the protein concentration in the stack.

Isotachophoresis on Polyacrylamide Gel

Abstract Isotachophoresis, using Ampholine spacer ions, was applied to the fractionation of two multicomponent protein systems (serum and a urinary preparation tract with Hunter Factor activity) using polyacrylamide gel as a supporting medium. These studies were designed to determine whether isotachophoresis could provide higher load capacity than obtained in conventional electrophoresis or isoelectric focusing on polyacrylamide gel without loss of resolution. The effect of the Ampholine pI range, the relative and absolute concentrations of protein sample and Ampholine, of the ionic strength of the gel buffer, and of the stacking limits obtained in various gel buffers was investigated. It was found that Ampholine components can act as spacers, giving rise to bands in what otherwise would be a continuum of stacked protein zones. Loads greater by 1 to 2 orders of magnitude than in conventional polyacrylamide gel electrophoresis were applicable. The resotion obtained was greatly inferior to that in PAGE, suggesting that Ampholine does not provide a sufficient distribution of constituent mobilities under the employed conditions. Isotachophoresis was applied at the preparative scale in two-stage fractionations designed to resolve multicomponent systems (serum).

Method of concentrating proteins on Sephadex prior to gel filtration

A simple method is described of stacking proteins on a Sephadex gel column by electrophoresis, followed by filtration through the column. The method has been applied to diluted blood sera, from which it appears that the proteins are concentrated even after gel filtration. The main advantage of the procedure is the elimination of other concentrating steps prior to gel filtration.