Gordon L. E. Koch

Perturbation of cellular calcium induces secretion of luminal ER proteins

The endoplasmic reticulum (ER) contains a family of luminal proteins (reticuloplasmins) that are normally excluded from the secretory pathway. However, reticuloplasmins are efficiently secreted when murine fibroblasts are treated with calcium ionophores. The secreted and cellular forms of endoplasmin are clearly distinguishable on the basis of gel mobility and endoglycosidase H sensitivity. Reticuloplasmin secretion leads to the depletion of the proteins from the ER and their accumulation in the Golgi apparatus. The stress response to calcium ionophore induces reaccumulation of reticuloplasmins in the ER and suppresses their secretion. Secretion is also associated with changes in the structure and distribution of the ER. These observations show that perturbation of cellular calcium levels leads to the breakdown of the mechanism for ER retention of reticuloplasmins and suggest a role for calcium ions in their sorting from secretory proteins.

The subunit structure of methionyl-tRNA synthetase from Escherichia coli

Methionyl-tRNA synthetase from E. coli has been studied quite extensively and several structural features of the enzyme have been defined. The native enzyme is purported to be a tetramer of identical subunits with molecular weights about 44000 each, Lemoine et al. [ 1 ] . An active tryptic fragment of this enzyme, which can be crystallised in a form well suited to studies by X-ray diffraction [2] has also been isolated. This fragment was thought to be a dimer of identical subunits with molecular weights of about 33000 each, formed by the removal of a segment of molecular weight 11000 from the native protomer [3] _ Another form of the enzyme has also been purified by Bruton and Hartley and shown to be a dimer of identical subunits with molecular weights of about 44000 [4]. The results described in this report show that the native enzyme is a homologous dimer, the protomers of which are actually about twice the molecular weight assigned previously and that the formation of the crystallisable fragment results from the removal of a segment of almost 200 residues from the C-terminal region of the native protomer.

Crystallization and preliminary X-ray diffraction studies on tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus☆

Abstract Conditions have been established for the crystallization of tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus at room temperature. The crystals are extremely well-ordered, exhibiting diffraction spots out to at least 2.7 A, and can be grown to a convenient size for X-ray crystallographic analysis. The crystals are trigonal with a space group P3121, the unit cell having dimensions of a = 64.4 A and c = 238 A ; the crystallographic asymmetric unit is probably one subunit of the dimeric (2 × 45,000, mol. wt) enzyme. The enzyme crystals are extremely stable and exhibit good resistance to radiation damage. This amino-acyl-tRNA synthetase appears to be amenable to complete structure determination by X-ray crystallography.

Identification of a novel calcium-binding protein (CP22) in multidrug-resistant murine and hamster cells

Analysis of cytoplasmic extracts of multidrug‐resistant murine and hamster cells by SDS gel and 2D gel electrophoresis showed that they expressed an abundant 22 kDa protein which was absent from the drugsensitive parent lines. SDS gel electrophoresis in the presence of EGTA and direct binding tests with 45Ca2+ showed that the resistance‐associated protein is a specific calcium‐binding protein. Thus the development of multidrug resistance in both colchicine‐selected hamster cells and adriamycin‐selected murine cells is associated with a major change in calcium metabolism. These observations provide the first molecular basis for the hypothesis that Ca2+ plays a central role in the development of the multidrug resistance phenomenon.

Repeated sequences in methionyl-tRNA synthetase from E. coli.

It has been argued that some of the diversity of the subunit structures and protomeric molecular weights of aminoacyl-tRNA synthetases is explained by repeated sequences in the larger protomeric units [ 11. The methionyl-, valyland leucyl-tRNA synthetases* from Bacillus stearothermophilus with protomeric molecular weights of 66 000,110 000 and 110 000 respectively were shown to contain long repeating sequences while the smaller tyrosyl-tRNA synthetase did not. Other reports, based on fingerprinting and peptide counting only, indicate that the isoleucyl[2] and leucyl[3] synthetases from E. coli with protomeric molecular weights of 102 000 and 100 000 respectively could have similar structural features. The methionyl-tRNA synthetase from E. coli has a native mol. wt. of about 175 000 [4,5] and a subunit size of 85 000 [6]. An active fragment of mol. wt. 65 000 can be made by proteolytic digestion [7] and this fragment has been shown to be a single polypeptide chain with the same amino-terminal sequence as the native enzyme [6]. Hence it must be made by the removal of about two hundred amino acids from the carboxyl-terminus of the native molecule which clearly results in the dissociation of the subunits. In this paper we show that, like the enzyme from B. stearothermophilus, MTS from E. coli contains a significiant amount of repeated sequences.

The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation.

Abstract The interaction of tRNATyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNATyr complex with [3H]acetic anhydride. In a second step the two 3H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [14C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their 14 C 3 H ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride. Involvement of lysine side-chains in complex formation with tRNATyr was suggested from altered 14 C 3 H ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNATyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNATyr complex has been detected. Only one molecule of tRNATyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.

Isolation and identification of partial cDNA clones for endoplasmin, the major glycoprotein of mammalian endoplasmic reticulum

The amino acid sequences of peptides isolated from murine endoplasmin showed significant homology (approximately 50%) with sequences in the heat-shock proteins 90 and 83 of yeast and Drosophila, respectively, indicating that they are related proteins. Mixed oligonucleotide probes, deduced from the peptide sequences, were used to isolate cDNAs from a murine liver cDNA library. DNA sequencing confirmed the presence of a coding sequence for one of the endoplasmin peptides, formally establishing the authenticity of the cDNA. The identity of the murine and hamster endoplasmin sequences suggests a level of sequence conservation associated with proteins that perform a structural role in cells.

An abundant ubiquitous glycoprotein (GP100) in nucleated mammalian cells.

Two‐dimensional gel electrophoresis with the 125I‐Con A overlay and affinity purification with Con A‐agarose revealed the presence of an abundant ubiquitous 100‐kDa glycoprotein (GP100) in nucleated mammalin cells. The amount in cultured human and murine cells varies from 3 to 20 × 106 molecules per cell making GP100 the most abundant glycoprotein in nucleated cells. Peptide mapping shows that it is different from erythrocyte Band III protein. Several properties of GP100 suggest that it could play a structural role in nucleated cell membranes.

Analysis of Pseudopodial Structure and Assembly with Viral Projections

SUMMARY The mechanisms by which cells extend motile pseudopodial projections are still poorly understood. Several fundamental mechanisms have been proposed on the basis of hydrostatic pressure, membrane addition and microfilament reorganization. A common focus of all such mechanisms is the growing tip of a pseudopodium. Yet some basic questions about the nature of the tip in natural pseudopodia remain obscure. However, one class of structure, the virus-tipped projections, often contains a well-defined particle, both morphologically and biochemically, and therefore provides a useful model system for the examination of the tips of cellular projections. In P815 cells the virus-tipped projections are long, thin structures closely resembling filopodia in other cells. The apical virus particle is a retrovirus particle produced by the chronic infection existing in this cell line. In demembranated filopodia, the virus particle retains a tight association with a single actin microfilament. Biochemical analyses indicate that the major retroviral structural polypeptide Pr65 is an actin-binding protein that could provide the anchorage site for the actin filament. The existence of a solid virus particle tethered by an actin filament to the cytoskeleton makes it very unlikely that these projections grow by membrane addition at the tip. The major positive implication is that the apex of a projection does not relinquish its interaction with the submembranous cytoskeleton during growth. Such an arrangement would be compatible with either a hydrostatic-pressure-driven or a cytoskeleton-driven mechanism of filopodial growth.

The Tryptophanyl- and Tyrosyl-tRNA Synthetases from Bacillus stearothermophilus

Aminoacyl-tRNA synthetases are ancient enzymes, as evidenced by their central role in metabolism (Granick 1950). They may well have evolved their specificities for amino acid and tRNA as the early cell developed pathways for amino acid synthesis and as the first codons were assigned to each amino acid (Wong 1975). Similarities between synthetases of different amino acid specificity might therefore reflect the mechanisms of earliest evolution. At first sight, the aminoacyl-tRNA synthetases seem to be a diverse group of enzymes with a range of quaternary structures ( α 1 , α 2 , and α 2 β 2 ) and subunit sizes (33,000–110,000 daltons) (see Table 1 of Hartley, this volume). Closer inspection reveals a loose class of large polypeptide chains centered on 100,000 daltons (and often monomeric) and a loose class of smaller chains centered on 50,000 daltons (and often dimeric). Peptide mapping, isolation of several tryptic peptides in greater than molar yield, and more detailed sequence studies have revealed that the large polypeptide chains contain areas of repeated sequence. (Kula 1973; Waterson and Konigsberg 1974; Koch et al. 1974). These larger chains could therefore have arisen by duplication and fusion of a common ancestral gene originally coding for the smaller chains. On the basis of this model, most synthetases would be composed of either a dimer of two identical chains or a monomer of fused domains. This arrangement would allow the binding of tRNA by contact of the pseudosymmetric regions on both tRNA and synthetase, each arm of the tRNA binding to corresponding portions of...