Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone,BiP

BiP, immunoglobulin binding protein, is an ER homologue of Hsp 70. However, unlike other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demonstrated the presence of potential regulatory proteins for BiP using a pull-down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull-down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein. Eight proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp 170 and BiP where identified, while the others were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for Bip was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.     

NMR structures of 36 and 73-residue fragments of the calreticulin P-domain

Calreticulin (CRT) is an abundant, soluble molecular chaperone of the endoplasmic reticulum. Similar to its membrane-bound homolog calnexin (CNX), it is a lectin that promotes the folding of proteins carrying N-linked glycans. Both proteins cooperate with an associated co-chaperone, the thiol-disulfide oxidoreductase ERp57. This enzyme catalyzes the formation of disulfide bonds in CNX and CRT-bound glycoprotein substrates. Previously, we solved the NMR structure of the central proline-rich P-domain of CRT comprising residues 189-288. This structure shows an extended hairpin topology, with three short anti-parallel beta-sheets, three small hydrophobic clusters, and one helical turn at the tip of the hairpin. We further demonstrated that the residues 225-251 at the tip of the CRT P-domain are involved in direct contacts with ERp57. Here, we show that the CRT P-domain fragment CRT(221-256) constitutes an autonomous folding unit, and has a structure highly similar to that of the corresponding region in CRT(189-288). Of the 36 residues present in CRT(221-256), 32 form a well-structured core, making this fragment one of the smallest known natural sequences to form a stable non-helical fold in the absence of disulfide bonds or tightly bound metal ions. CRT(221-256) comprises all the residues of the intact P-domain that were shown to interact with ERp57. Isothermal titration microcalorimetry (ITC) now showed affinity of this fragment for ERp57 similar to that of the intact P-domain, demonstrating that CRT(221-256) may be used as a low molecular mass mimic of CRT for further investigations of the interaction with ERp57. We also solved the NMR structure of the 73-residue fragment CRT(189-261), in which the tip of the hairpin and the first beta-sheet are well structured, but the residues 189-213 are disordered, presumably due to lack of stabilizing interactions across the hairpin.     

Dimerization,ubiquitylation and endocytosis go together in growth hormone receptor function

Internalization of membrane proteins has been studied for more than three decades without solving all the underlying mechanisms. Our knowledge of the clathrin-coated endocytosis is sufficient to understand the basic principles. However, more detailed insight is required to recognize why different proteins enter clathrin-coated pits with different rates and affinities. In addition to clathrin coat components, several adapter systems and even more accessory proteins have been described to preselect membrane proteins before they can enter cells. Recent experimental data have identified the ubiquitin-proteasome system as a regulatory system both in endocytic and lysosomal membrane traffic. This system is well-known for its basic regulatory function in protein degradation, and controls a magnitude of key events. In this review, we will discuss the complexity and implications of this mechanism for membrane trafficking with emphasis on the growth hormone receptor.     

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.     

Automated recognition of intracellular organelles in confocal microscope images

Recognition of the localisation of intracellular proteins is essential to the understanding of their function. It is usually made through knowledge of and comparison to the distribution of well-characterised intracellular organelles by experts in cell biology. We have automated this process in order to achieve a more objective and quantitative assessment of the protein distribution within the cell, which can be employed by the less experienced cell biologist and may be utilised as a training program for inexperienced users, or as a high throughput localisation program for novel genes in functional analysis. Here we describe the development and testing of a classification system based on a modular neural network trained with sets of confocal sections through cell lines fluorescently stained for markers of key intracellular structures. The system functioned well in spite of the variability in pattern that occurs between individual cells and performed with 97% accuracy, which gives us confidence in the method and in its future development. It is envisaged that this program will aid the design of further experiments utilising colocalisation with known organelle marker proteins, in order to confirm putative trafficking pathways and protein–protein interactions of the protein of interest.     

Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca²⁺) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca²⁺ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca²⁺ uptake of +33%. Specific Ca²⁺ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca²⁺ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca²⁺ re-uptake function of the SR under defined culture conditions.     

Changes in the levels of seven proteins involved in polypeptide folding and transport during endosperm development of two barley genotypes differing in storage protein localisation

The Russian barley cultivar Nevsky lacks ₃ hordein and accumulates most of its hordein in the lumen of the endoplasmic reticulum and only a minor portion in the vacuole. In wild type barley and all other temperate cereals, storage proteins are deposited in the vacuole. F1 crosses revealed that the Nevsky phenotype is recessive; but the extent of hordein accumulation in the endoplasmic reticulum in F2 endosperm lacking ₃ hordein was very much less than in the Nevsky parent. In order to study the Nevsky endosperm phenotype we have measured the levels of seven proteins and two mRNAs involved in protein folding in the ER lumen or ER to Golgi transport during endosperm development. The protein levels were unaltered in Nevsky as compared to the wild-type variety Bomi. When the levels of these seven proteins were correlated with the rate of hordein accumulation, four of these (HSP70, PDI, Sar1p and Sec18p) were consistently up-regulated with hordein synthesis. Accumulation of hordein in the endoplasmic reticulum appears to be determined by the absence of ₃ hordein, or the product of a gene closely linked to it, plus one or more other recessive genes.     

Relationship between development of endosperm transfer cells and grain mass in maize

The most basal endosperm cells of maize (Zea mays L.) began differentiating into transfer cells in 10 days after pollination (DAP). The thickening and ingrowths forming in the transfer cell wall were slow during 10 and 15 DAP. There were many vesicles, silky and string ball objects in cytoplasm, and the number of mitochondria and rough endoplasm reticulum increased. After 15 DAP, the wall thickening and ingrowths forming in the transfer cells sped up. By 20 DAP, the transfer cell zone had developed, there appeared 65 - 70 rows of cells in width and 3 - 4 layers of cell in depth, the obvious cell wall ingrowths presented strong positive reaction with periodic acid Schiff's reagent. After 20 DAP, no significant change appeared in the shape and structure of the transfer cells, and the transfer cells entered function stage. In the mature kernels (53 DAP), the most basal transfer cells were filled with ingrowths, however, dense cytoplasm was also found in these cells. The nuclei had quite irregular shapes in these cells. Some transfer cells contained black grains and crystals. A black layer formed in the pericarp tissue adjacent to the transfer cell zone. Full development of endosperm transfer cells was important for reduction of kernel abortion and increase of kernel mass. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.