Yeast two-hybrid assay for examining human immunodeficiency virus protease heterodimer formation with dominant-negative inhibitors and multidrug-resistant variants.

The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HIV) protease (PR) monomers. Defective monomers that were previously shown to exhibit a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay. Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AIDS patient were tested for interaction in yeast. Dimer formation between wt monomers with catalytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demonstrated. The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for the probing of interactions between monomers with mdr-associated mutations with those based on the HIV-1 HXB2R sequence. The HTLVIII/HIV-1 HXB2R clone has been the basis for a large number of HIV-related plasmids, primers, antibodies, and other specific reagents throughout the HIV research community. The results of our assay suggest that HXB2R-based D-N PR inhibitors associate with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus. These results further encourage the use of D-N PR inhibitors as antiviral agents which may complement existing small-molecule combination therapies.

Protein disulfide isomerase in spore germination and cell division.

Protein disulfide isomerase (PDI) is a protein of the endoplasmic reticulum (ER) that is essential for the unscrambling of nonnative disulfide bonds. Here, we have determined the importance of PDI to both spore germination and vegetative cell division. To vary the concentration of PDI in the ER, we used plasmids that direct the expression of rat PDI fused at its N terminus to either the alpha-factor pre-pro segment or the alpha-factor pre sequence, and fused at its C terminus to either the mammalian (KDEL) or the yeast (HDEL) ER retention signal. Classical yeast genetic (tetrad) analyses, and plasmid loss and plasmid shuffling experiments were used to evaluate the ability of these constructs to complement haploid Saccharomyces cerevisiae cells in which the endogenous PDI1 gene had been deleted. We find that basal levels of PDI in the ER are sufficient for vegetative growth. In contrast, high levels of PDI in the ER are required for efficient spore germination. Thus, catalysis of the unscrambling of nonnative disulfide bonds in cellular proteins is more important during spore germination than during vegetative cell division.

The CXXC motif: imperatives for the formation of native disulfide bonds in the cell.

The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active-site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.

The essential function of protein-disulfide isomerase is to unscramble non-native disulfide bonds.

Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC. Haploid pdi1 delta Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease). In contrast, pdi1 delta yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction. SGHC PDI catalyzes none of these processes. These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.

Production of rat protein disulfide isomerase in Saccharomyces cerevisiae.

Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and reduction of protein disulfide bonds. Saccharomyces cerevisiae cells lacking PDI are inviable. PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear. A cDNA that codes for rat PDI fused to the alpha-factor pre-pro segment was expressed in a protease-deficient strain of S. cerevisiae under the control of an ADH2-GAPDH hybrid promoter. The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus. The typical yield of isolated protein was 2 mg per liter of culture. The catalytic activity of the PDI from S. cerevisiae was indistinguishable from that of PDI isolated from bovine liver. This expression system is unique in allowing the same plasmid to be used both to complement pdi1 delta S. cerevisiae and to produce PDI for detailed in vitro analyses. Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.