Import into and Degradation of Cytosolic Proteins by Isolated Yeast Vacuoles

In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet

The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an l-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, l-[1-13C]cysteine was given as a primed, constant i.v. infusion (3μmol⋅kg−1⋅h−1) for 6 h, and incorporation of label into whole blood GSH determined by GC/MS selected ion monitoring. The fractional synthesis rate (mean ± SD; day-1) of whole blood GSH was 0.65 ± 0.13 for the adequate diet and 0.49 ± 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 ± 243 and 1,216 ± 162 μM for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 ± 216 and 579 ± 135 μmol⋅liter−1⋅day−1, respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

The chemistry of the S-nitrosoglutathione/glutathione system

S-Nitrosothiols have generated considerable interest due to their ability to act as nitric oxide (NO) donors and due to their possible involvement in bioregulatory systems—e.g., NO transfer reactions. Elucidation of the reaction pathways involved in the modification of the thiol group by S-nitrosothiols is important for understanding the role of S-nitroso compounds in vivo. The modification of glutathione (GSH) in the presence of S-nitrosoglutathione (GSNO) was examined as a model reaction. Incubation of GSNO (1 mM) with GSH at various concentrations (1–10 mM) in phosphate buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous oxide, and ammonia as end products. The product yields were dependent on the concentrations of GSH and oxygen. Transient signals corresponding to GSH conjugates, which increased by one mass unit when the reaction was carried out with 15N-labeled GSNO, were identified by electrospray ionization mass spectrometry. When morpholine was present in the reaction system, N-nitrosomorpholine was formed. Increasing concentrations of either phosphate or GSH led to lower yields of N-nitrosomorpholine. The inhibitory effect of phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N2O3), formed by oxidation of NO. This supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for virtually all of the GSNO nitrogen consumed during the reaction, and it is now possible to construct a complete set of pathways for the complex transformations arising from GSNO + GSH.

Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with different in vivo half-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+ T cells.

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s−1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.

Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis

Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis. In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed “supernatant protein factor (SPF),” which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid [Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921–930]. Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF. Here we report the cDNA cloning and expression of SPF from rat and human. The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as α-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein. Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro. SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis. SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture. Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas. Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis. These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67PHOX and cytochrome b558

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac1 or Rac2, with the membrane-bound cytochrome b558. Whereas the interaction of p47PHOX with cytochrome b558 is well established, an interaction between p67PHOX and cytochrome b558 has never been investigated. We report here a direct interaction between p67PHOX and cytochrome b558. First, labeled p67PHOX recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b558-deficient patient. Second, p67PHOX binds to cytochrome b558 that has been bound to nitrocellulose. Third, GTP-p67PHOX bound to glutathione agarose is able to pull down cytochrome b558. Rac1-GTP or Rac1-GDP increased the binding of p67PHOX to cytochrome b558, suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67PHOX and cytochrome b558.

Signaling Mediated by the Cytosolic Domain of Peptidylglycine α-Amidating Monooxygenase

The luminal domains of membrane peptidylglycine α-amidating monooxygenase (PAM) are essential for peptide α-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.