Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The plasma membrane H+-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H+-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae, even after deletion of the enzyme's carboxy terminus. Functional chimerical H+-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H+-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H+-ATPase. The reverse Ala-598-->Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H+-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K(m) values for MgATP and decreased Vmax compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H+-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.

Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H(+)-ATPases. We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H(+)-ATPase isoform of Nicotiana plumbaginifolia. Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted. The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0. The N. plumbaginifolia PMA2 expressed in S. cerevisiae has unusual low Km for ATP (23 microM) and high pH optimum (6.8). Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium. Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker. These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions.

Characterization of the HBsAg particle lipid membrane.

HBsAg particles are highly immunogenic and have been shown to be a suitable support for the presentation of foreign epitopes. More information about the topology of the HBsAg protein is a prerequisite to any rational attempt to replace the region of this protein with foreign epitopes without modifying the assembly of the particles. This topology and, more precisely, the mode of interaction of the HBsAg protein with the lipid will depend on the lipid organization in the particle envelope. Nothing is known concerning the lipid organization of HBsAg particles. The only available information concerns their lipid composition. Phospholipase D hydrolysis of HBsAg particles was used here to determine whether the particles were surrounded with a lipid monolayer or bilayer. The lipid fluidity within the particle envelope was evaluated by fluorescence polarization measurements. The data strongly suggest that the HBsAg particle membrane is organized as a discontinuous rigid bilayer of lipids interacting with protein aggregates.

Overexpression in Escherichia coli and purification of an ATP-binding peptide from the yeast plasma membrane H(+)-ATPase.

The two major hydrophilic domains from the Saccharomyces cerevisiae plasma membrane H(+)-ATPase fused to glutathione S-transferase have been expressed in Escherichia coli. The GST-L peptide contained the hydrophilic region from Ala340 to Ser660. The GST-SL peptide contained in addition the hydrophilic region Glu162 to Val276. After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatography, 3 mg of highly purified peptides were recovered per liter of E. coli culture. The purified peptides interacted with 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which was enhanced identically upon binding of either GST-L or GST-SL. ATP competitively displaced the TNP-ATP binding. The observed dissociation constants for TNP-ATP (6.5 microM) and ATP (3 mM) are close to those found for the complete native H(+)-ATPase protein. The fluorescence of TNP-ATP was sensitive to Mg2+ indicating the existence of a Mg(2+)-binding site on the peptide. Apparent affinity for this Mg2+ site was found to vary from 50 microM at pH 7.5 to 400 microM at pH 5.5.

Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae.

OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. METHODS: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement.

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells.

The unprocessed Gag precursor from HIV-1, when expressed in recombinant baculovirus-infected insect cells, is targeted to the plasma membrane and assembles in 100-120 nm particles budding from the cell surface. This process mimics HIV immature particle formation and is dependent on myristoylation of the N-terminal glycine, as deletion of the latter results in particle accumulation in the cytoplasm and, interestingly, in the nucleus, pointing to a potential role of this non-fatty-acid-acylated species in the viral life cycle. Inclusion of the pol gene in the construct results in efficient processing of Pr55gag and a pronounced decrease in particle formation. Deletion of the C terminus (p16) of the Gag precursor, including the finger domains, abolishes particle assembly, but membrane targeting and evagination still occur. Heterologous expression in insect cells may prove very useful for the study of the molecular events leading to retroviral particle morphogenesis.

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.

The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane.

Myristoylation of the Pr65gag protein from Moloney murine leukemia virus has been shown to be essential for virus particle formation [Rein et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7246-7250], and by analogy, myristoylation of the human immunodeficiency virus (HIV) Gag precursor could possibly play a similar role. We have investigated the expression and myristoylation of the complete HIV Gag precursor Pr55gag in yeast, the subcellular localization of that protein, and the contribution of the myristoyl-glycine residue to this localization. Immunogold labelling of myristoylated Pr55gage with antibodies directed against HIV Gag products was apparent in the vicinity of the plasma membrane. On the contrary, non-myristoylated derivatives of Pr55gag were only detected in relatively well-defined regions of the cytoplasm. These results show that targeting and accumulation of the HIV Gag precursor, Pr55gag, at the plasma membrane occurs in yeast in the absence of other viral components and requires the N-myristoyl-glycine residue.