Intravacuolar membrane lysis in Saccharomyces cerevisiae. Does vacuolar targeting of Cvt17/Aut5p affect its function?

The integral membrane protein Cvt17/Aut5p is a putative lipase essential for intravacuolar lysis of autophagic bodies. It is localized at the endoplasmic reticulum, from which it is targeted via the multivesicular body (MVB) pathway to intravacuolar MVB vesicles. Proteinase protection experiments now demonstrate that the Aut5 amino terminus is located in the cytosol, and the carboxyl terminus is located inside the ER lumen. In contrast to procarboxypeptidase S, targeting of Cvt17/Aut5p to MVB vesicles is not blocked in cells lacking the ubiquitin ligase Tul1p or the deubiquitinating enzyme Doa4p. Also, truncation of the amino-terminal cytosolic Cvt17/Aut5p domain does not inhibit its targeting to MVB vesicles. These findings suggest that similar to Sna3p sorting of Cvt17/Aut5p to MVB vesicles is independent of ubiquitination. By fusing the ER retention/retrieval signal HDEL to the carboxyl terminus of Cvt17/Aut5p, we generated a construct that is held back at the ER. Detailed analysis of this construct suggests an essential role of vacuolar targeting of Cvt17/Aut5p for its function. Consistently, aut5Delta cells are found impaired in vacuolar degradation of autophagocytosed peroxisomes. Importantly, biochemical and morphological data further suggest involvement of Cvt17/Aut5p in disintegration of intravacuolar MVB vesicles. This points to a general function of Cvt17/Aut5p in intravacuolar membrane breakdown.     

Vipera lebetina venom contains two disintegrins inhibiting laminin-binding beta1 integrins

To explain the myotoxic effects of snake venoms, we searched for inhibitors of alpha7beta1 integrin, the major laminin-binding integrin in skeletal muscle. We discovered two inhibitors in the venom of Vipera lebetina. One of them, lebein-1 (known as lebein), has already been proposed to be a disintegrin because of its RGD-containing primary sequence. The other, lebein-2, is a novel protein that also interacts firmly with alpha3beta1, alpha6beta1, and alpha7beta1 integrins, but not with the collagen-binding alpha1beta1 and alpha2beta1 integrins. Ligand binding of laminin-recognizing beta1 integrins was efficiently blocked by both lebein-1 and lebein-2. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, neither lebein-1 nor lebein-2 interacted with alpha7beta1 integrin in an RGD-dependent manner, similar to the interaction of the laminin with alpha7beta1 integrin. Identical divalent cation dependence of integrin binding to laminin and to either of the two inhibitors and their mutually exclusive binding suggest that both lebein-1 and lebein-2 interact with the ligand-binding site of laminin-binding beta1 integrins by mimicking the yet unknown integrin-binding structure of laminins. Like lebein-1, lebein-2 is a soluble heterodimeric disintegrin of low molecular mass. Together with membrane-bound ADAM-2 and ADAM-9, the two inhibitors seem to form a small group of disintegrins that can bind to laminin-binding beta1 integrins. Because of their inhibitory capability both in vitro and in vivo, lebein-1 and lebein-2 may be valuable tools in influencing laminin-induced, integrin-mediated cell functions such as cell anchorage, migration, and mechanical force transduction on laminin-rich basement membranes.     

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.     

Early detection in saliva of polypeptides associated to isoproterenol-induced mouse parotid hypertrophy

Chronic administration of isoproterenol (IPR) results in a marked hypertrophy and in the induction of a group of putative proline-rich polypeptides in the mouse parotid glands. Some of these polypeptides (pps C-G) have been considered as molecular markers of the parotid gland enlargement. Given the secretory character of polypeptides C-G, the polypeptide composition of mouse saliva was used to monitor the IPR-induced salivary gland hypertrophy. Whole saliva was collected after an oral administration of pilocarpine (PIL). Under those conditions, PIL provoked a massive salivary secretion both in normal control mice and during the whole course of the IPR-induced gland enlargement. Striking changes in the polypeptide composition of saliva obtained from chronically IPR-stimulated animals were observed. Those changes consisted basically in the appearance and progressive increase in concentration of parotid polypeptides C-G and in the progressive diminution in concentration of a couple of normal salivary polypeptides (polypeptides A-B). The appearance of new polypeptides in saliva could be established unequivocally within the 24 h following the trophic adrenergic stimulation. On the other hand, salivary polypeptides induced in response to a single administration of IPR could be demonstrated as late as 7-9 days after the stimulation. Accordingly, detection of parotid polypeptides C-G in PIL-produced saliva obtained from IPR-stimulated mice has proved to be a highly advantageous method to evaluate salivary gland hypertrophy both at very early stages after the trophic stimulation and late after the occurrence of the trophic episode.     

Modulation of nuclear pore topology by transport modifiers

The nuclear pore complex (NPC) represents the only pathway for macromolecular communication between the nuclear and cytoplasmic compartments of the cell. Nucleocytoplasmic transport requires the interaction of transport receptors with phenylalanine-glycine (FG)-repeats that line the transport pathway through the NPC. Here we examine the effects of transport receptors and amphipathic alcohols on NPC topology using scanning force microscopy. We show that transport receptors that irreversibly bind FG-repeats increase NPC vertical aspect, whereas transport receptors that weakly interact with FG-repeats increase NPC diameter. Interestingly, small polar alcohols likewise increase NPC diameter. These opposing effects agree with the inhibition or enhancement of nuclear transport, respectively, previously ascribed to these agents.     

Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly

Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine-less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)-dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface.     

Signal peptide of Lassa virus glycoprotein GP-C exhibits an unusual length

Lassa virus glycoprotein is synthesized as precursor GP-C into the lumen of the endoplasmic reticulum and cleaved posttranslationally into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by subtilase SKI-1/S1P. The N-terminal portion of the primary translation product preGP-C contains a signal peptide of unknown length. In order to demonstrate the signal peptide cleavage site, purified viral GP-1 isolated from Lassa virus particles was N-terminally sequenced as TSLYKGV, identical to amino acids 59-65 of GP-C. Mutational analysis of the amino acid residues flanking the putative cleavage site led to non-cleavable preGP-C indicating that no other signal peptide cleavage site exists. Interestingly, GP-C mutants with a non-cleavable signal peptide were not further processed by SKI-1/S1P. This observation suggests that the signal peptide cleavage is necessary for GP-C maturation and hence for Lassa virus replication.     

11th International Congress on Molecular Plant-Microbe Interactions held at St. Petersburg--a report

The report is a short summary of the most interesting presentations at the 11th International Congress on Molecular Plant-Microbe Interactions held during July 18-27, 2003 at St. Petersburg, Russia. The key elements from several sessions on the legume-Rhizobium interactions have been discussed.     

Requirements for Cu(A) and Cu-S center assembly of nitrous oxide reductase deduced from complete periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida

Bacterial nitrous oxide (N(2)O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N(2)O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu(A) site in the periplasm. There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco. We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida. An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N(2)O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N(2)O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu(A) metallation. Both of these were found to be dispensable elements for N(2)O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N(2)O reductase maturation.