Architecture of coatomer: Molecular characterization of delta-COP and protein interactions within the complex

Copyright © 2011 by The Rockefeller University Press.Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.This work was supported by The Deutsche Forschungsgemeinschaft (SFB 352), the Human Frontier Science Program, and the Swiss National Science Foundation No. 31-43366.95

Relation of Toxicity and Conformation of Phallotoxins as Revealed by Optical Rotatory Dispersion Studies

Natural variants and chemically modified derivatives of phalloidin have been found or prepared which show all degrees of toxicity. High toxicity persists if only three of the side chains are modified. Acetylation of two of the four OH groups of phalloidin leads to less toxic products, the triacetyl derivative is nontoxic. On tosylation toxicity disappeared after reaction of only two OH groups. Alkylation of the indole nitrogen leads to equally or less toxic (N-methy), -ethyl, carbamoylmethyl, -tert.butyl) or nontoxic (N-propyl, -pentyl) products. Toxicity is also completely absent if only one of the two rings of the molecule is intact. In optical rotatory dispersion spectra all of the toxic compounds show in aqueous solution two cotton effects centered around 300 nm and 248 nm, whereas in nearly all of the nontoxic derivatives at least one of the effects is missing. Triacetylphalloidin and tribromophalloidin, both nontoxic, also exhibit “toxic” curves in water. Their conformation, however, changes on going to organic solvents like methanol or acetonitril as observed by optical rotatory dispersion studies. Thus, stability of the “aqueous” conformation in a lipophilic environment is apparently the second prerequisite for a toxic action of phallopeptides

Syntheses of further analouges of norphalloin. Gly1-, L.Val1- and D-Abu2-Norphalloin and (β-Trideutero)-Ala5-Norphalloin.

The norphalloin analogues with glycine and L-valine instead of L-alanine in position 1, (II and III), with D-α-aminobutyric acid instead of D-threonine in position 2 (IV), and with β-trideutero-L-alanine instead of L-alanine in position 5 (β-D3-I), have been synthesized according to Chart I, mainly using the mixed anhydride method. For the final cyclization step to III the p-nitrophenylester method was employed. Analogue IV showed toxicity in the white mouse with doses <5 mg per kg body weight, whereas II and III were nontoxic. The deuterated norphalloin analogue proved by p.m.r. measurement that in fact the methyl group of alanyl residue no.5 is located above the indole nucleus, as suggested in a former publication (4)

Reduced phallotoxin uptake by livers of young compared with adult rats

Using [3H]-demethylphalloin as a tracer the uptake of phallotoxins by the liver of young (6, 12, 16 days old) and adult rats was determined in relation to the dose of toxin administered. The maximum amount taken up by the livers of the young rats was only about 50% of that in adults. Nevertheless, with a dose as high as 55 mg/kg body weight the toxin concentration in the young liver reached more than 30 micrograms/g, being markedly higher than the minimum concentration (approximately 20 micrograms/g) required to cause irreversible damage of the liver in adult rats and death of the animals. This suggests that the tolerance of young rats to phallotoxins cannot solely be explained by the reduced uptake of the toxin