cDNA cloning of a human 100 kDa de-ubiquitinating enzyme: the 100 kDa human de-ubiquitinase belongs to the ubiquitin C-terminal hydrolase family 2 (UCH2).

The full length cDNA encoding a 100 kDa human de-ubiquitinating enzyme, referred to as de-ubiquitinase was obtained using one clone selected from a randomly sequenced human brain cDNA library and specific primers. The sequence of 18 peptides generated from the de-ubiquitinase isolated from out-dated human erythrocytes matched perfectly with the predicted amino acid sequence, which would encode a protein containing 858 amino acids (calculated M(r) = 95,743 Da). Homology search disclosed that the protein is a member of a large family of ubiquitin C-terminal hydrolases (UCH2), that was defined on the basis of the presence of two specific patterns, 'the Cys- and His-domains', which are likely to be involved in the de-ubiquitinating activity [7]. An additional conserved region, 'the aspartic acid domain', was also identified, the functional role of which is unknown.

A human de-ubiquitinating enzyme with both isopeptidase and peptidase activities in vitro.

Some enzymatic and physicochemical properties of a human ubiquitin-specific isopeptidase are reported. The enzyme was purified to homogeneity from red blood cells and its specificity towards polymeric ubiquitin substrates suggests a de-ubiquitinating activity capable of cleaving 'head-to-tail' polyUb chains as well as isoamide 'branched' Ub dimers. KM values show a 10 fold preference for the cleavage of branched Ub dimers over head-to-tail Ub dimers. The enzymatic activity can be strongly inhibited by various peptides containing either of the cleavage site sequences found in Ub polymers, but not by unrelated peptides. The enzyme is monomeric under reducing conditions and exhibits a globular shape with an average diameter of 9 nm, an S20,w value of 5.2 S and a molar mass of 110 kDa +/- 10%. Because the enzyme cleaves both peptide-linked and isopeptide-linked Ub moieties from substrates, we propose to name it de-ubiquitinase rather than isopeptidase.

Secretion of an Mr 60000 protein by benomyl-treated cells of Neurospora crassa.

In the presence of the microtubule inhibitor benomyl at micron concentrations, cells of Neurospora crassa wild type strain St. Lawrence 74A were found to secrete high amounts of an Mr 60 000 protein into the culture medium (about 35 micrograms/ml after a 12 h treatment). The secretion also occurred after treatment with the other antitubulin drugs carbendazim (MBC), nocodazole, thiabendazole, and griseofulvin. This secretion is apparently induced by the specific action of benomyl on N. crassa beta-tubulin as no secretion of the Mr 60 000 protein could be detected after treatment of the benomyl-resistant mutant bml 511 (r), mutated in its beta-tubulin gene (Orbach et al., Mol. Cell. Biol. 6, 2452-2461 (1986)). The secretion was abolished by 12 microM cycloheximide, a protein synthesis inhibitor. The Mr 60 000 protein could be separated into two main and four secondary components by two-dimensional gel electrophoresis (pI 6.67 and 6.52 and pI 6.93, 6.81, 6.44, and 6.32, respectively). The Mr 60 000 protein was not a major intracellular protein of benomyl-treated cells and could only be revealed by immunoblotting with polyclonal antibodies raised against the extracellular form. It was undetectable in untreated cells collected at various stages of vegetative growth or in their culture medium.

Isolation and characterization of coated vesicles from filamentous fungi.

Coated vesicles have been shown to exist in Neurospora crassa (Ascomycetes) and Uromyces phaseoli (Basidiomycetes) growing germlings. Separation of coated vesicles in both fungi was obtained when the high-speed (100,000g) pellet was fractioned on a Sephacryl S-1000 gel filtration column, according to the procedure of Mueller and Branton. Electron micrographs of negatively stained coated vesicles from fractions of gel filtration show the same striking lattice coated vesicles similar to vertebrate coated vesicles. We observe two major size classes of coated vesicles in both fungi: the larger class (100-180 nm) is similar in size to vertebrate coated vesicles; the smaller class (50-80 nm) is mostly found in both fungi. When examined by SDS-PAGE, the Sephacryl column fractions containing the maximum concentration of electron microscopically visible coated vesicles coincide with the bands of the protein coat reported as clathrin. The protein composition on SDS-PAGE of the coated vesicles indicates a major polypeptide species of 180 kDa and minor 30 to 36 kDa species. Polypeptides of 100 kDa and 64 kDa are also found in the fractions containing coated vesicles.