Cytosolic Protein Factor Research Articles

Abstract P3-06-12: Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?

Abstract Proteasome, the essential giant protease, is the hub of the ubiquitin-proteasome pathway critical for maintaining intracellular proteostasis. Inhibitors of the proteasome cause overload of cells with non-degraded protein debris and activate proapoptotic signaling. The effects are especially dramatic in fast metabolizing cancer cells; hence the proteasome inhibitors are used as anti-cancer drugs. There are two FDA-approved drugs directly blocking the proteasome activity, bortezomib and carfilzomib, and several others are in trials. So far, the high effectiveness of these drugs is limited to multiple myeloma and other hematological malignancies. Unfortunately, breast and other solid cancers are much more resistant to apoptosis triggered by attenuation of the ubiquitin-proteasome pathway than majority of blood cancers. The relatively low effectiveness of proteasome inhibitors has been noted even for cancers that have been identified in genome-wide screens as addicted to the proteasome activity, such as triple negative breast cancers. The source of the resistance, noted in cell culture, animal studies and clinical trials, remains unknown. Here we propose that, at least in part, the resistance can be tracked down to protein-protein interactions between the drug target, the proteasome, and a cytosolic heat shock protein, Hsp72 (inducible Hsp70). Indeed, high levels of Hsp72 have been observed in many cancers, including breast, and generally correlate with poor prognosis. The intracellular roles of the ubiquitous chaperone are undoubtedly very diverse, however we postulate a novel molecular action: direct protection of the proteasome from competitive inhibitors of its enzymatic activity. The hypothesis has been inspired by our recent work exploring the very effective ubiquitin-proteasome pathway in the naked mole rats. These small African rodents do not develop cancers and maintain unusually long lifespan and health-span. Interestingly, the proteasome in naked mole rats is exceptionally resistant to inhibition. We discovered that the resistance is bestowed by a cytosolic protein factor containing Hsp72 and Hsp40 chaperones (Biochim. Biophys. Acta-MBD 1842; 2060-2072; 2014). We partially purified the factor and tested its in vitro effectiveness with human proteasomes. The molecular mechanism beneficial for naked mole rats may be also protective for human cancer cells. We used pifithrin μ, a specific small molecule inhibitor blocking the interactions of Hsp72 with other proteins, to test the hypothesis. We prepared lysates from cultured triple negative breast cancer cells MDA-MB-231. Such lysates are rich in proteasome activity and preserve interactions of the proteasomes with natural protein ligands. Interestingly, upon the treatment with pifithrin μ proteasomes in lysates maintained their high activity while becoming significantly more sensitive to inhibition with bortezomib. The studies are in progress to assess the identity of a putative chaperone-based proteasome resistance factor in breast cancer cells. Citation Format: Gaczynska M, Osmulski PA. Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-12.

A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition

The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31years) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although Heat shock protein 72 kDa (HSP72) and Heat shock protein 40 kDa (Homolog of bacterial DNAJ1) (HSP40(Hdj1)) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging.

The in Vitro RNA Synthesizing Activity of the Isolated Arterivirus Replication/Transcription Complex Is Dependent on a Host Factor

The cytoplasmic replication of positive-stranded RNA viruses is associated with characteristic, virus-induced membrane structures that are derived from host cell organelles. We used the prototype arterivirus, equine arteritis virus (EAV), to gain insight into the structure and function of the replication/transcription complex (RTC) of nidoviruses. RTCs were isolated from EAV-infected cells, and their activity was studied using a newly developed in vitro assay for viral RNA synthesis, which reproduced the synthesis of both viral genome and subgenomic mRNAs. A detailed characterization of this system and its reaction products is described. RTCs isolated from cytoplasmic extracts by differential centrifugation were inactive unless supplemented with a cytosolic host protein factor, which, according to subsequent size fractionation analysis, has a molecular mass in the range of 59-70 kDa. This host factor was found to be present in a wide variety of eukaryotes. Several EAV replicase subunits cosedimented with newly made viral RNA in a heavy membrane fraction that contained all RNA-dependent RNA polymerase activity. This fraction contained the characteristic double membrane vesicles (DMVs) that were previously implicated in EAV RNA synthesis and could be immunolabeled for EAV nonstructural proteins (nsps). Replicase subunits directly involved in viral RNA synthesis (nsp9 and nsp10) or DMV formation (nsp2 and nsp3) exclusively cosedimented with the active RTC. Subgenomic mRNAs appeared to be released from the complex, whereas newly made genomic RNA remained more tightly associated. Taken together, our data strongly support a link between DMVs and the RNA-synthesizing machinery of arteriviruses.

The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCζ

When sperm activate eggs at fertilization the signal for activation involves increases in the intracellular free Ca2+ concentration. In mammals the Ca2+ changes at fertilization consist of intracellular Ca2+ oscillations that are driven by the generation of inositol 1,4,5-trisphosphate (InsP3). It is not established how sperm trigger the increases in InsP3 and Ca2+ at fertilization. One theory suggests that sperm initiate signals to activate the egg by introducing a specific factor into the egg cytoplasm after membrane fusion. This theory has been mainly based upon the observation that injecting a cytosolic sperm protein factor into eggs can trigger the same pattern of Ca2+ oscillations induced by the sperm. We have recently shown that this soluble sperm factor protein is a novel form of phospholipase C (PLC), and it is referred to as PLCzeta(zeta). We describe the evidence that led to the identification of PLCzeta and discuss the issues relating to its potential role in fertilization.

Teleost fish spermatozoa contain a cytosolic protein factor that induces calcium release in sea urchin egg homogenates and triggers calcium oscillations when injected into mouse oocytes

Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCγ). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCζ). Homologues of PLCζ have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.

Flowering Plant Sperm Contains a Cytosolic Soluble Protein Factor Which Can Trigger Calcium Oscillations in Mouse Eggs

There is evidence showing that the sperm-induced Ca2+ oscillations in mammalian eggs at fertilization are triggered by a sperm-derived protein factor. It was established recently that the activity of the putative sperm protein in causing Ca2+ oscillations in mammalian eggs is not species-specific in vertebrates (1, 16). Here we report that cytosolic soluble extracts derived from flowering plant sperms in Brassica campestris can also induce fertilization-like Ca2+ oscillations when microinjected into mouse eggs. The factor responsible for inducing Ca2+ oscillations in the plant sperm was sperm-specific and heat- or trypsin-labile. Eight to ten sperm equivalents of the plant sperm extracts had enough activity to trigger Ca2+ oscillations in mouse eggs. Our study suggests that, although plant and mammal are evolutionary divergent species, the activity of the putative sperm protein factor in triggering Ca2+ signaling in mammalian eggs is not specific to the animal kingdom.

Enhancement of Transport-dependent Decarboxylation of Phosphatidylserine by S100B Protein in Permeabilized Chinese Hamster Ovary Cells

Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.

A Microsomal GTPase Is Required for Glycopeptide Export from the Mammalian Endoplasmic Reticulum

Bidirectional transport of proteins via the Sec61p translocon across the endoplasmic reticulum (ER) membrane is a recognized component of the ER quality control machinery. Following translocation and engagement by the luminal quality control system, misfolded and unassembled proteins are exported from the ER lumen back to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and glycopeptides, are efficiently exported from mammalian and yeast systems, indicating that bidirectional transport across ER membranes is a general eukaryotic phenomenon. Glycopeptide and protein export from the ER in in vitro systems is both ATP- and cytosol-dependent. Using a well established system to study glycopeptide export and conventional liquid chromatography, we isolated a single polypeptide species of 23 kDa from rat liver cytosol that was capable of fully supporting glycopeptide export from rat microsomes in the presence of an ATP-regenerating system. The protein was identified by mass spectrometric sequence analysis as guanylate kinase (GK), a housekeeping enzyme critical in the regulation of cellular GTP levels. We confirmed the ability of GK to substitute for complete cytosol by reconstitution of glycopeptide export from rat liver microsomes using highly purified recombinant GK from Saccharomyces cerevisiae. Most significantly, we found that the GK (and hence the cytosolic component) requirement was fully bypassed by low micromolar concentrations of GDP or GTP. Similarly, export was inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a requirement for GTP hydrolysis. Membrane integrity was fully maintained under assay conditions, as no ER luminal proteins were released. Competence for glycopeptide export was abolished by very mild protease treatment of microsomes, indicating the presence of an essential protein on the cytosolic face of the ER membrane. These data demonstrate that export of glycopeptide export is controlled by a microsomal GTPase and is independent of cytosolic protein factors.

The soluble mammalian sperm factor protein that triggers Ca 2+ oscillations in eggs: Evidence for expression of mRNA(s) coding for sperm factor protein(s) in spermatogenic cells

At fertilisation in mammals the sperm initiates a series of Ca 2+ oscillations that activate development. One theory of signalling at fertilisation suggests that the sperm contains a soluble protein factor that causes these Ca 2+ oscillations by entering the egg after sperm–egg membrane fusion. This theory is supported by the finding that, in some species, injection of sperm protein extracts into eggs triggers a pattern of Ca 2+ oscillations similar to those seen at fertilisation. So far, all the direct evidence for a sperm factor has been based upon the injection of soluble proteins from mature sperm. Here, we demonstrate that injection of mRNA extracted from hamster spermatogenic cells also leads to generation of prolonged Ca 2+ oscillations in mouse eggs. The ability of spermatogenic cell mRNA to induce Ca 2+ oscillations is dependent upon translation into protein and also appears to be specific to spermatogenic cells since injection of mRNA isolated from somatic tissues into eggs was ineffective. These data support the hypothesis that sperm contain a soluble, cytosolic protein factor that induces Ca 2+ oscillations in eggs at fertilisation. These data are discussed in the light of our recent findings that suggest that the sperm factor possesses a phospholipase C activity.

Ca(2+) oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs.

At fertilization in mammals, the sperm activates the egg by inducing a series of oscillations in the intracellular free Ca(2+) concentration. There is evidence showing that this oscillatory event is triggered by a sperm-derived protein factor which diffuses into egg cytoplasm after gamete membrane fusion. At present the identity of this factor and its precise mechanism of action is unknown. Here, we studied the specificity of action of the sperm factor in triggering Ca(2+) oscillations in mammalian eggs. In doing so, we examined the patterns of Ca(2+) signaling in mouse eggs, zygotes, parthenogenetic eggs and maturing oocytes following the stimulation of bovine sperm extracts which contain the sperm factor. It is observed that the sperm factor could induce Ca(2+) oscillations in metaphase eggs, maturing oocytes and parthenogenetically activated eggs but not in the zygotes. We present evidence that Ca(2+) oscillations induced by the sperm factor require a maternal machinery. This machinery functions only once in mammalian oocytes and eggs, and is inactivated by sperm-derived components but not by parthenogenetic activation. In addition, it is found that neither InsP(3) receptor sensitivity to InsP(3) nor Ca(2+) pool size are the determinants that cause the fertilized egg to lose its ability to generate sperm-factor-induced Ca(2+) oscillations at metaphase. In conclusion, our study suggests that the orderly sequence of Ca(2+) oscillations in mammalian eggs at fertilization is critically dependent upon the presence of a functional maternal machinery that determines whether the sperm-factor-induced Ca(2+) oscillations can persist.

Xenopus and Chicken Sperm Contain a Cytosolic Soluble Protein Factor Which Can Trigger Calcium Oscillations in Mouse Eggs

There is evidence showing that at fertilization the sperm introduces into egg cytoplasm a protein-based cytosolic factor, which serves as the physiological trigger for inducing Ca2+ oscillations in mammalian eggs. Here we show that sperm of nonmammalian vertebrates also contain a cytosolic protein factor that can induce Ca2+ oscillations when introduced into mammalian eggs. We have observed that cytosolic extracts derived from Xenopus or chicken sperm could induce mouse eggs to undergo Ca2+ oscillations similar to those induced by bovine sperm extracts. The factor responsible for inducing Ca2+ oscillations was of high molecular weight and heat- or proteinase K-labile. We show that 0.5 chicken sperm-equivalents or 1–2 Xenopus sperm-equivalents of the extracts had enough activity to trigger Ca2+ oscillations in mouse eggs. Our findings illustrate that although Xenopus, chicken, and mammals are evolutionarily divergent species, the function of the sperm protein factor in triggering Ca2+ oscillations in mammalian eggs appears not to be species specific in vertebrates.

Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis

Hydrogenosomes are double membrane bounded redox organelles found in a number of amitochondriate protists and fungi. They are involved in carbohydrate metabolism and ATP synthesis and thus resemble mitochondria. Molecular analysis of the hydrogenosomal heat shock proteins Hsp70, Hsp60 and Hsp10 in Trichomonas vaginalis, one of the deepest-branching eukaryotes known to date, has revealed that these group exclusively with mitochondrial heat shock proteins. This finding indicates strongly that a progenitor organelle which gave rise to contemporary mitochondria and hydrogenosomes existed early in eukaryotic life. This hypothesis is further supported by similarities of hydrogenosomal and mitochondrial biogenesis. It was shown that T. vaginalis hydrogenosomal proteins are synthesized on free ribosomes in the cytosol with an N-terminal presequence that carries targeting information and is cleaved upon import into the organelle. Furthermore, as in mitochondrial import, hydrogenosomal protein import requires ATP, an electrochemical transmembrane potential and cytosolic protein factor(s). Here we demonstrate that inhibition of hydrogenosomal protein import occurs (i) in the presence of a synthetic presequence peptide and (ii) after pretreatment of hydrogenosomes with the protease trypsin. Trypsin pretreatment affects two hydrogenosomal membrane proteins of 31 and 70 kDa, respectively. Thus, we present evidence that import is saturable and that proteinaceous hydrogenosomal import receptor(s) exist. These results are a first step towards a characterization of the hydrogenosomal import machinery which should provide further insights into the relationship of hydrogenosomes and mitochondria and the evolution of protein targeting into organelles of endosymbiotic origin.

Post-transcriptional Regulation of H-ferritin mRNA: IDENTIFICATION OF A PYRIMIDINE-RICH SEQUENCE IN THE 3′-UNTRANSLATED REGION ASSOCIATED WITH MESSAGE STABILITY IN HUMAN MONOCYTIC THP-1 CELLS

We have previously demonstrated that phorbol myristate acetate (PMA) up-regulates H-ferritin gene expression in myeloid cells by stabilization of its message. In the present report, we showed that insertion of the 3'-untranslated region (3'-UTR) of H-ferritin mRNA at the 3'-end of luciferase coding sequence significantly reduced the stability of luciferase mRNA in human monocytic THP-1 cells. However, the half-life of the chimeric transcript was markedly prolonged after PMA treatment. A cytosolic protein factor from THP-1 cells was found to specifically bind to H-ferritin 3'-UTR. PMA treatment of THP-1 cells resulted in the reduction of the RNA binding activity in a time-dependent manner. Deletion analysis and RNase T1 mapping revealed a pyrimidine-rich sequence within the 3'-UTR which interacts with the protein factor. Competition experiments with homoribopolymers further demonstrated the importance of uridines for the binding activity. Point mutations in uridines of the pyrimidine-rich sequence reduced the protein binding to 3'-UTR, while increasing the stability of the chimeric luciferase transcript. Together, these results demonstrate that the pyrimidine-rich sequence in the 3'-UTR is involved in post-transcriptional regulation of H-ferritin gene expression in myeloid cells.

Injection of sperm extract mimics spatiotemporal dynamics of Ca2+ responses and progression of meiosis at fertilization of ascidian oocytes.

Sperm extract (SE) of the ascidian, Ciona savignyi, injected into oocytes induced repetitive intracellular Ca2+ increases with kinetics consistent with those at fertilization and caused reinitiation and progression of meiosis as in fertilized oocytes with the formation of polar bodies. The Ca2+ response comprised two sets of Ca2+ oscillations separated by 5 minutes and correlated with the first and second meiotic metaphase. The effects of SE were dose dependent and the critical dose corresponded roughly to a single spermatozoon. In the first Ca2+ transient observed by confocal microscopy, a Ca2+ wave started from the SE injection site at the peripheral region of the oocyte and propagated across the ooplasm. The similar wave was produced by injection at the central region, starting from an arbitrary cortical area after 30 seconds, probably after SE had diffused to the cortex. The sensitivity to SE is thought to be preferentially higher in the cortex. The effective component of SE was heat-unstable, and its molecular weight was estimated as in the range between 10x10(4 )and 3x10(4) using membrane filters. These results suggest that, in ascidian fertilization, a cytosolic sperm protein factor is introduced to the oocyte cortex and induces Ca2+ waves and thereby meiotic resumption, leading to cell-cycle-correlated Ca2+ oscillations.

Protein Folding and Transport from the Endoplasmic Reticulum to the Golgi Apparatus in Plants

Summary Protein folding and transport in the plant secretory pathway is a new and rapidly growing area of research. The results obtained so far have revealed a high degree of conservation of the proteins involved between plants, yeast and mammals, and therefore tools and techniques developed for yeast and mammalian systems have helped in the elucidation of the plant secretory pathway. Newly synthesised proteins are folded in the lumen of the endoplasmic reticulum (ER) and here they acquire their secondary, tertiary and sometimes quaternary structure. The folding process is not spontaneous, but is helped by a set of ER resident proteins called chaperones. These transiendy associate with the newly synthesised polypeptide chain and help folding and prevent misfolding. Once a protein is correctly folded, it travels along the secretory pathway to its final destination. Transport from the ER to the next step, the Golgi apparatus, is vesicle-mediated, requiring a set of cytosolic proteins called coat proteins (COPs) and energy in the form of sugar triphosphates. Similarly, fusion with the cis-Golgi is dependent on cytosolic protein factors and energy. This review summarizes what is known about protein folding and ER to Golgi transport in plants.

A Cytosolic Sperm Protein Factor Mobilizes Ca2+ from Intracellular Stores by Activating Multiple Ca2+ Release Mechanisms Independently of Low Molecular Weight Messengers

Ca2+ oscillations can be induced in mammalian eggs and somatic cells by microinjection of a cytosolic sperm protein factor. The nature of the sperm factor-induced Ca2+ signaling was investigated by adding sperm protein extracts to homogenates of sea urchin eggs, which contain multiple classes of Ca2+ release mechanisms. We show that the sperm factor mobilizes Ca2+ from non-mitochondrial Ca2+ stores in egg homogenates after a distinct latency. This latency is abolished by preincubation of sperm extracts with egg cytosol. The preincubation step is highly temperature-dependent and generates a high molecular weight, protein-based Ca2+-releasing agent that can also mobilize Ca2+ from purified egg microsomes. This Ca2+ release appears to be mediated via both inositol 1,4,5-trisphosphate and ryanodine receptors, since homologous desensitization of these two release mechanisms by their respective agonists inhibits further release by the sperm factor. However, sperm factor-induced Ca2+ release by these channels is independent of inositol 1,4, 5-trisphosphate or cADPR since antagonists of either of these two messengers did not block the Ca2+ release effected by the sperm factor. The sperm protein factor may cause Ca2+ release via an enzymatic step that generates a protein-based Ca2+-releasing agent.

Cytosolic factors in mitochondrial protein import.

In vitro import studies have confirmed the participation of cytosolic protein factors in the import of various precursor proteins into mitochondria. The requirement for extramitochondrial adenosine triphosphate for the import of a group of precursor proteins seems to be correlated with the chaperone activity of the cytosolic protein factors. One of the cytosolic protein factors is hsp70, which generally recognizes and binds unfolded proteins in the cytoplasm. Hsp70 keeps the newly synthesized mitochondrial precursor proteins in import-competent unfolded conformations. Another cytosolic protein factor that has been characterized is mitochondrial import stimulation factor (MSF), which seems to be specific to mitochondrial precursor proteins. MSF recognizes the mitochondrial precursor proteins, forms a complex with them and targets them to the receptors on the outer surface of mitochondria.

Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.

To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radio-labeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of p alpha F was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.

A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes.

Previously it has been shown that injecting a cytosolic sperm protein factor into mammalian eggs induces sustained repetitive transients of cytosolic free Ca2+ ([Ca2+]i), or [Ca2+]i oscillations [Swann (1990) Development 110, 1295-1302]. These sperm-factor (SF)-induced [Ca2+]i oscillations are similar to those seen at fertilization. Here we demonstrate that injecting the same cytosolic extracts of mammalian sperm into single rat hepatocytes induces a series of [Ca2+]i oscillations, as measured by aequorin luminescence. SF injection into hepatocytes induced [Ca2+]i oscillations that were of longer duration, lower frequency and greater amplitude than those seen with the Ins (1,4,5)P3-generating agonist phenylephrine. The SF-induced [Ca2+]i responses appeared to be due to internal release of Ca2+, since transients could occur in Ca(2+)-free media. Addition of the phorbol ester phorbol 12,13-dibutyrate (PDBu) at low concentrations did not inhibit the SF-induced [Ca2+]i oscillations; high concentrations of PDBu led to a sustained increase in [Ca2+]i concentrations. These data demonstrate that sperm contain a protein factor capable of inducing a characteristic series of [Ca2+]i oscillations in a somatic cell, the hepatocyte. Along with previous observations in dorsal root ganglion neurons, the data suggest a widespread efficacy of the factor in triggering Ca2+ oscillations.

Mechanisms of tRNA import into yeast mitochondria: an overview.

Mitochondrial import of tRNA is now considered as a quasi-universal phenomenon. In the yeast Saccharomyces cerevisiae, one of the three lysine isoacceptors, the tRNA(Lys)1 with the anticodon CUU (tRNA-K1), is encoded by the nuclear genome and distributed between the cytoplasmic (> 95%) and mitochondrial (< 5%) compartments. In vivo and in vitro import assays were developed to study the mechanisms of tRNA-K1 mitochondrial import. Transmembrane translocation of the tRNA requires the intactness of at least two of the components of the mitochondrial import machinery of pre-proteins, MOM19 and MIM44, as well as energy of ATP hydrolysis and an electrochemical potential across the inner membrane. The import of tRNA-K1 involves formation of an RNP complex on the mitochondrial outer membrane. tRNA-K1 import is also dependent upon cytosolic protein factors, one of which was identified as the precursor of the mitochondrial lysyl-tRNA synthetase (MSK). Although essential for tRNA-K1 import in vitro and in vivo, pre-MSK is however not sufficient to direct the import in vitro, which suggests the need of additional cytosolic factor(s). The tRNA can be imported in its mature form and nucleoside modification is not essential. Aminoacylation of the imported tRNA by the cytoplasmic lysyl-tRNA synthetase is a prerequisite for import. Possible mechanisms of intracellular partitioning and mitochondrial membrane translocation of tRNA-K1 are discussed.