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Knowing the molecular makeup of an organ system is required for its in-depth understanding. We analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance our knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless ( btl ) -Gal4 is not able to target all parts of the adult tracheal system. Here, we have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system.

Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity. Hijacking host cell processes applies in particular to the hepatitis B virus (HBV), as its DNA genome with about 3 kb in size is one of the smallest viral genomes known. HBV is a leading cause of liver disease and still displays one of the most successful pathogens in human populations worldwide. The extremely successful spread of this virus is explained by its efficient transmission strategies and its versatile particle types, including virions, empty envelopes, naked capsids, and others. HBV exploits distinct host trafficking machineries to assemble and release its particle types including nucleocytoplasmic shuttling transport, secretory, and exocytic pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Understanding how HBV uses and subverts host membrane trafficking systems offers the chance of obtaining new mechanistic insights into the regulation and function of this essential cellular processes. It can also help to identify potential targets for antiviral interventions. Here, I will provide an overview of HBV maturation, assembly, and budding, with a focus on recent advances, and will point out areas where questions remain that can benefit from future studies. Unless otherwise indicated, almost all presented knowledge was gained from cell culture-based, HBV in vitro-replication and in vitro-infection systems., (© 2022 The Authors. Biology of the Cell published by Wiley-VCH GmbH on behalf of Société Française des Microscopies and Société de Biologie Cellulaire de France.)

Several decades after its discovery, the hepatitis B virus (HBV) still displays one of the most successful pathogens in human populations worldwide. The identification and characterization of interactions between cellular and pathogenic components are essential for the development of antiviral treatments. Due to its small-sized genome, HBV highly depends on cellular functions to produce and export progeny particles. Deploying biochemical-silencing methods and molecular interaction studies in HBV-expressing liver cells, we herein identified the cellular ERGIC-53, a high-mannose-specific lectin, and distinct components of the endoplasmic reticulum (ER) export machinery COPII as crucial factors of viral trafficking and egress. Whereas the COPII subunits Sec24A, Sec23B and Sar1 are needed for both viral and subviral HBV particle exit, ERGIC-53 appears as an exclusive element of viral particle propagation, therefore interacting with the N146-glycan of the HBV envelope in a productive manner. Cell-imaging studies pointed to ER-derived, subcellular compartments where HBV assembly initiates. Moreover, our findings provide evidence that HBV exploits the functions of ERGIC-53 and Sec24A after the envelopment of nucleocapsids at these compartments in conjunction with endosomal sorting complexes required for transport (ESCRT) components. These data reveal novel insights into HBV assembly and trafficking, illustrating therapeutic prospects for intervening with the viral life cycle.

Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host-pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N-terminal half of Sec24A and a di-arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross-talk might display a tool to effectively inhibit SVP release., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Hepatitis B virus (HBV) is a leading cause of liver disease and is presently estimated to infect more than 250 million humans. The extremely successful spread of this virus among the human population is explained by its effective transmission strategies and its manifold particle types, including virions, empty envelopes and naked capsids. Due to its tiny genome, HBV depends on cellular machineries to thrive in infected hepatocytes. To enter, traverse and exit the cell, HBV exploits host membrane trafficking pathways, including intracellular highways directed by Rab GTPases. Here, we review recent discoveries focused on how HBV co-opts and perturbs host Rab GTPase functions with an emphasis on Rab7A- and Rab33B-mediated trafficking pathways. Rab7A plays bidirectional roles in the viral life cycle, as it promotes the endocytic uptake of HBV in early stages, but restricts exocytic virion release in late stages. In intermediate stages of HBV propagation, Rab33B is needed to guide the assembly of replicative progeny nucleocapsids. Rab33B acts together with its Atg5-12/16L1 effector, a protein complex required for autophagosome formation, suggesting the concept that HBV exploits this Rab/effector complex as an assembly scaffold and machine. We also discuss whether Rab-directed trafficking pathways engaged by HBV may be applicable to other virus families. Identification of overlapping Rab functions may offer new chances to develop broad-spectrum host-targeted antiviral strategies.

Chronic obstructive pulmonary disease (COPD) is among the most important causes of death. Signaling systems that are relevant for tissue repair and detoxification of reactive oxygen species or xenobiotics are thought to be impaired in lungs of patients suffering from this disease. Here, we developed a simple cigarette smoke induced Drosophila model of COPD based on chronic cigarette smoke exposure that recapitulates major pathological hallmarks of the disease and thus can be used to investigate new therapeutic strategies. Chronic cigarette smoke exposure led to premature death of the animals and induced a set of phenotypes reminiscent of those seen in COPD patients, including reduced physical activity, reduced body fat, increased metabolic rate and a substantial reduction of the respiratory surface. A detailed transcriptomic analysis revealed that especially the TGF-β, Nrf2 and the JAK/STAT signaling pathways are altered by chronic cigarette smoke exposure. Based on these results, we focused on Nrf2 signaling. A pharmacological intervention study performed with oltipraz, an activator of Nrf2 signaling, increased survival of cigarette smoke exposed animals significantly. Thus, the Drosophila COPD model recapitulates many major hallmarks of COPD and it is highly useful to evaluate the potential of alternative therapeutic strategies.

Previous studies indicated that hepatitis B virus (HBV) stimulates autophagy to favor its production. To understand how HBV co-opts autophagy as a proviral machinery, we studied the roles of key autophagy proteins in HBV-replicating liver cell cultures. RNA interference-mediated silencing of Atg5, Atg12, and Atg16L1, which promote autophagophore expansion and LC3 membrane conjugation, interfered with viral core/nucleocapsid (NC) formation/stability and strongly diminished virus yields. Concomitantly, the core/NC membrane association and their sorting to envelope-positive compartments were perturbed. A close inspection of the HBV/autophagy cross talk revealed that the virus depended on Atg12 covalently conjugated to Atg5. In support of this finding, HBV required the E2-like enzymes Atg10 and Atg3, which catalyze or facilitate Atg5-12 conjugation, respectively. Atg10 and Atg3 knockdowns decreased HBV production, while Atg3 overexpression increased virus yields. Mapping analyses demonstrated that the HBV core protein encountered the Atg5-12/16L1 complex via interaction with the intrinsically disordered region of the Atg12 moiety that is dispensable for autophagy function. The role of Atg12 in HBV replication was confirmed by its incorporation into virions. Although the Atg5-12/16L1 complex and Atg3 are essential for LC3 lipidation and, thus, for autophagosome maturation and closure, HBV propagation did not require LC3. Silencing of LC3B, the most abundant LC3 isoform, did not inhibit but rather augmented virus production. Similar augmenting effects were obtained upon overexpression of a dominant negative mutant of Atg4B that blocked the lipid conjugation of the LC3 isoforms and their GABARAP paralogues. Together, our data indicate that HBV subverts early, nondegradative autophagy components as assembly scaffolds, thereby concurrently avoiding autophagosomal destruction. IMPORTANCE Infections with the hepatitis B virus (HBV), an enveloped pararetrovirus, cause about 1 million deaths per year, as current therapies rarely achieve a cure. Understanding the HBV life cycle and concomitant host cell interactions is instrumental to develop new antiviral concepts. Here, we proceeded to dissect the roles of the autophagy machinery in virus propagation. By using RNA interference and overexpression studies in HBV-replicating cell lines, we identified the autophagic Atg5-12/16L1 elongation complex along with Atg10 and Atg3 to be an essential scaffold for HBV nucleocapsid assembly/stability. Deficits in Atg5-12/16L1 and Atg10/Atg3, which normally drive autophagophore membrane expansion, strongly impaired progeny virus yields. HBV gained access to Atg5-12/16L1 via interaction of its core protein with the Atg12 moiety of the complex. In contrast, subsequent autophagosome maturation and closure events were unnecessary for HBV replication, as evidenced by inhibition of Atg8/LC3 conjugation. Interfering with the HBV/Atg12 cross talk may be a tool for virus control., (Copyright © 2018 American Society for Microbiology.)

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites., Competing Interests: The authors declare no conflict of interest.

The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements.

Hepatitis B virus morphogenesis is accompanied by the production and release of non-enveloped capsids/nucleocapsids. Capsid particles are formed inside the cell cytosol by multimerization of core protein subunits and ultimately exported in an uncommon coatless state. Here, we investigated potential roles of Rab GTPases in capsid formation and trafficking by using RNA interference and overexpression studies. Naked capsid release does not require functions of the endosome-associated Rab5, Rab7 and Rab27 proteins, but depends on functional Rab33B, a GTPase participating in autophagosome formation via interaction with the Atg5-Atg12/Atg16L1 complex. During capsid formation, Rab33B acts in conjunction with its effector, as silencing of Atg5, Atg12 and Atg16L1 also impaired capsid egress. Analysis of capsid maturation steps revealed that Rab33B and Atg5/12/16L1 are required for proper particle assembly and/or stability. In support, the capsid protein was found to interact with Atg5 and colocalize with Atg5/12/16L1, implicating that autophagy pathway functions are involved in capsid biogenesis. However, a complete and functional autophagy pathway is dispensable for capsid release, as judged by knockdown analysis of Atg8/LC3 family members and pharmaceutical ablation of canonical autophagy. Experiments aimed at analysing the capsid release-stimulating activity of the Alix protein provide further evidence for a link between capsid formation and autophagy., (© 2014 John Wiley & Sons Ltd.)

The hepatitis B virus (HBV) is an enveloped DNA virus that replicates via reverse transcription of its pregenomic RNA (pgRNA). Budding of HBV is supposed to occur at intracellular membranes and requires scission functions of the endosomal sorting complex required for transport (ESCRT) provided by ESCRT-III and VPS4. Here, we have investigated the impact of the upstream-acting ESCRT-I and ESCRT-II complexes in HBV morphogenesis. RNA interference knockdown of the ESCRT-I subunits TSG101 and VPS28 did not block, but rather stimulate virus release. In contrast, RNAi-mediated depletion of the ESCRT-II components EAP20, EAP30 and EAP45 greatly reduced virus egress. By analyzing different steps of the HBV maturation pathway, we find that the knockdown of ESCRT-II not only inhibited the production and/or release of enveloped virions, but also impaired intracellular nucleocapsid formation. Transcription/translation studies revealed that the depletion of ESCRT-II neither affected the synthesis and nuclear export of HBV-specific RNAs nor the expression of the viral core and envelope proteins. Moreover, the absence of ESCRT-II had no effects on the assembly capability and integrity of HBV core/capsids. However, the level of encapsidated pgRNA was significantly reduced in ESCRT-II-depleted cells, implicating that ESCRT-II directs steps accompanying the formation of replication-competent nucleocapsids, like e.g. assisting in RNA trafficking and encapsidation. In support of this, the capsid protein was found to interact and colocalize with ESCRT-II subunits in virus-producing cells. Together, these results indicate an essential role for ESCRT-II in the HBV life cycle and suggest that ESCRT-II functions prior to the final HBV budding reaction.

Muscodor albus, a biofumigant fungus, has the potential to control post-harvest pathogens in storage. It has been shown to produce over 20 volatile compounds with fungicidal, bactericidal and insecticidal properties. However, M. albus is a warm climate endophyte, and its biofumigant activity is significantly inhibited at temperatures below 5 °C. Conidia of seven mycotoxin producing fungi, Aspergillus carbonarius, A. flavus, A. niger, A. ochraceus, Penicillium verrucosum, Fusarium culmorum and F. graminearum, were killed or prevented from germinating by exposure to volatiles from 2 g M. albus-colonized rye grain per L of headspace in sealed glass jars for 24 h at 20 °C. Two major volatiles of M. albus, isobutyric acid (IBA) and 2-methyl-1-butanol (2MB) at 50 µL/L and 100 µL/L, respectively, gave differential control of the seven fungi when applied individually at 20 °C. When the fungi were exposed to both IBA and 2MB together, an average of 94% of the conidia were killed or suppressed. In a factorial experiment with controlled atmosphere storage (CA) at 3 °C and 72 h exposure to four concentrations of IBA and 2MB combinations, 50 µL/L IBA plus 100 µL/L 2MB killed or suppressed germination of the conidia of all seven fungi. Controlled atmosphere had no significant effect on conidial viability or volatile efficacy. Major volatiles of M. albus may have significant potential to control plant pathogens in either ambient air or CA storage at temperatures below 5 °C. However, combinations of volatiles may be required to provide a broader spectrum of control than individual volatiles.

Hepatitis B virus (HBV) is a major cause of liver disease. Due to the tiny size of its genome, HBV depends on the critical interplay between viral and host factors for the generation of new viral particles from infected cells. Recent work has illuminated a multiplicity of spatially and temporally coordinated virus-host interactions that accompany HBV particle genesis. These interactions include the requirement of cellular chaperones for the maturation of the three viral envelope proteins, the cellular factors involved in dynamic modification, maturation, and intracellular trafficking of the nucleocapsids, and the host components of the multivesicular body (MVB) pathway enabling virion budding at intracellular compartments. Beside infectious virions, HBV produces at least two other types of particles, subviral empty envelope particles and subviral naked capsid particles, likely as a result of the engagement of different host factors by the viral structural proteins. Accordingly, HBV exploits distinct cellular pathways to release its particle types. Here, I review recent progress in these areas of the cell biology of HBV genesis.

The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning ER reporters are not affected. Consistent with this, the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levels independent of its Sec62-interacting motif, indicating that it may not act in conjunction with Sec62 in human cells. The specific action of Sec63 is further sustained by our observations that the up-regulation of either Sec62 or two other ER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. A J domain-specific mutation of Sec63, proposed to weaken its interaction with the ER resident BiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these results suggest that Sec63 may perform a substrate-selective quantity control function during cotranslational ER import.

Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT., (© 2010 Blackwell Publishing Ltd.)

γ2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that γ2-adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit γ2-adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed γ2-adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess γ2-adaptin acts by accelerating lysosomal Gag destruction. Consistently, depletion of γ2-adaptin avoids Gag routing to the lysosome and increases viral production. Functional studies with natural MVB cargoes support a role of γ2-adaptin in MVB-to-lysosome transition. Furthermore, we show that different members of the endosomal sorting complex required for transport (ESCRT) that drive sorting from endosomes to lysosomes are sequestered upon γ2-adaptin overexpression. If sequestered irreversibly, they are targeted to enhanced lysosomal degradation. The participation of γ2-adaptin in MVB sorting is further suggested by our finding that it specifically interacts with the ESCRT subunits Vps28 and CHMP2A. These observations identify γ2-adaptin as a critical factor in MVB trafficking, which likely is involved in endosome-to-lysosome maturation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-adaptin. By mapping regions of Nedd4 involved in binding to gamma2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-adaptin. In addition, we show that gamma2-adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-adaptin, a closely related homolog of gamma2-adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-adaptin may operate within the MVB sorting system in a manner different from that of classic adaptins.

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.

The hepatitis B virus L protein forms a dual topology in the endoplasmic reticulum (ER) via a process involving cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain. Here, we show that preS posttranslocation depends on the action of the ER chaperone BiP. To modulate the in vivo BiP activity, we designed an approach based on overexpressing its positive and negative regulators, ER-localized DnaJ-domain containing protein 4 (ERdj4) and BiP-associated protein (BAP), respectively. The feasibility of this approach was confirmed by demonstrating that BAP, but not ERdj4, destabilizes the L/BiP complex. Overexpressing BAP or ERdj4 inhibits preS posttranslocation as does the reduction of ATP levels. These results hint to a new role of BiP in guiding posttranslational polypeptide import into the mammalian ER.

Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor gamma 2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role gamma 2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess gamma 2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed gamma 2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of gamma 2-adaptin.

Background: The addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. Here, we report on an exception to this rule occurring on the hepatitis B virus (HBV) large L envelope protein that is a subject to co-plus posttranslational N-glycosylation., Results: By using an improved detection system, we identified so far unrecognized, novel isoforms of L. Based on mutational analyses, the use of N-glycosylation inhibitors, and pulse-chase studies, we showed that these isoforms are due to posttranslational N-glycan addition to the asparagines 4 and 112 within the preS domain of L. While an inhibition of N-glycosylation and glycan trimming profoundly blocked virus assembly and release, the posttranslational N-glycosylation of L itself was found to be dispensable for HBV morphogenesis., Conclusion: These data together with previous results implicate that the N-glycosylation requirements of virion release are due to functional inhibition of cell glycoproteins engaged by HBV.

Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular gamma2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered gamma2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same gamma2-adaptor and that disruption of the HBV/gamma2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of gamma2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both gamma2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with gamma2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.

The envelope of hepatitis B virus (HBV), containing the L, M, and S proteins, is essential for virus entry and maturation. For direct visualization of HBV, we determined whether envelope assembly could accommodate the green fluorescent protein (GFP). While the C-terminal addition of GFP to S trans-dominant negatively inhibited empty envelope particle secretion, the N-terminal GFP fusion to S (GFP.S) was co-integrated into the envelope, giving rise to fluorescent particles. Microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of GFP.S, required for particle export were rescued by interprotein interactions with wild-type S. Thereby, a dual location of GFP, inside and outside the envelope, was observed. GFP.S was also efficiently packaged into the viral envelope, and these GFP-tagged virions retained the capacity for attachment to HBV receptor-positive cells in vitro. Together, GFP-tagged virions should be suitable to monitor HBV uptake and egress in live hepatocytes.

During the life cycle of hepatitis B virus (HBV) the large L envelope protein plays a pivotal role that is related to its peculiar dual transmembrane topology. To study the complex structure and diverse functions of L under regulated conditions of production, a human 293 cell line stably expressing L under the control of the ecdysone-inducible promoter was generated. Cells demonstrated stringent dose- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer. Temporal control of L expression allowed to trace (i) its posttranslational reorientation resulting in the mixed topology; (ii) its spatial redistribution from the endoplasmic reticulum to Golgi-like structures; and (iii) its intracellular retention in a membrane-associated configuration. On regulated overproduction, L blocked the secretion of HBV small envelope polypeptides without impairing the cell secretory apparatus. Despite the continuous high-level storage of L within the 293 cell line, no cytopathic effects could be detected. This is in contrast to ground-glass hepatocytes of chronic HBV carriers and HBV transgenic mice and may imply that the intracellular storage of L is particularly damaging to the liver cell.

For functional diversity, the large (L) envelope protein of hepatitis B virus (HBV) acquires a dual transmembrane topology via co-translational membrane integration of the S region and partial post-translational translocation of the preS subdomain. Because each process requires the second transmembrane segment (TM2), we explored the action of this determinant by using protease protection analysis of mutant L proteins. We demonstrated that neither the disruption of a leucine zipper-like motif by multiple alanine substitutions nor the flanking charges of TM2 affected the topological reorientation of L. The dispensability of both putative subunit interaction modules argues against a link between preS post-translocation and envelope assembly. Phenotypic mixing experiments revealed that the preS and S protein domains of the related duck HBV L polypeptide failed to substitute functionally for the topogenic elements of HBV in directing the correct L topogenesis, implicating different translocation mechanisms used by the two hepadnavirus genera.

Liver damage caused by chronic hepatitis B virus (HBV) infection may be enhanced through the selection of deleted HBV preS mutants by intracellular accumulation of viral proteins and subsequent cell death. However, the prevalence and impact of such mutants on the clinical course of infection have not yet been studied in children. Serum samples from 60 children (mean age 9.8 y) were investigated by means of PCR and direct sequencing of the entire preS region. Only one patient (1.5%) was found with a mixed HBV population of a deletion spanning 183 nucleotides and wild-type sequences. This mutation alters the HBV large-surface protein and removes the small-surface promoter. To clarify the significance of this mutation, we studied 14 serial serum samples of the child within a follow-up of 10 y. After occurrence of the mutation, the liver enzymes increased, despite seroconversion to anti-HBe. Transfection of an HBV expression construct containing this deletion into human hepatoma cells by using an HBV in vitro replication system showed that the mutant lost the ability of nucleocapsid packaging as a result of alteration of the transmembrane topology of the large surface protein. This effect could not be restored by coexpression of wild-type large- or small-surface proteins in trans. In conclusion, the circulation of HBV preS deletion mutants is rare in childhood. However, our functional and clinical follow-up studies in one child suggest that such a mutant may have the potential to aggravate liver inflammation, especially if corroborated with larger numbers of children.

The large L envelope protein of the hepatitis B virus utilizes a new folding pathway to acquire a dual transmembrane topology in the endoplasmic reticulum (ER). The process involves cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain into the ER. Here, we demonstrate that the conformational and functional heterogeneity of L depends on the action of molecular chaperones. Using coimmunoprecipitation, we observed specific interactions between L and the cytosolic Hsc70, in conjunction with Hsp40, and between L and the ER-resident BiP in mammalian cells. Complex formation between L and Hsc70 was abolished when preS translocation was artificially switched to a cotranslational mode, implicating Hsc70 to act as a preS holding and folding catalyst that controls partial preS posttranslocation. The functional role of Hsc70 in L topogenesis was confirmed through modulation of its in vivo activity by overexpressing its co-chaperones Hip and Bag-1. Overexpression of the Hsc70-stimulating molecule Hip led to increased entrapping of preS on the cytosolic ER face and hence to a decrease in preS posttranslocation, whereas the negative regulator Bag-1 had the opposite effects. Furthermore, Hip-mediated Hsc70 activation impaired virus production in hepatitis B virus-replicating hepatoma cells, likely due to the improper topological reorientation of L. Together, these results indicate that translocational regulation of protein topology by chaperones provides a means of generating structural and functional diversity. They also hint to the dynamic nature of the mammalian ER translocation machinery in handling co- and posttranslational substrates.

The large (L) envelope protein of the hepatitis B virus (HBV) has the peculiar capacity to form two transmembrane topologies via an as yet uncharacterized process of partial post-translational translocation of its pre-S domain across membranes. In view of a current model that predicts an HBV-specific channel generated during virion envelope assembly to enable pre-S translocation, we have examined parameters influencing L topogenesis by using protease protection analysis of wild-type and mutant L proteins synthesized in transfected cells. We demonstrate that contrary to expectation, all determinants, thought to be responsible for channel formation, are dispensable for pre-S reorientation. In particular, we observed that this process does not require (i) the helper function of the HBV S (small) and M (middle) envelope proteins, (ii) covalent dimer formation of envelope chains, or (iii) either of the three amphipathic transmembrane segments of L. Rather, the most hydrophobic transmembrane segment 2 of L was identified as a vital topogenic determinant, essential and sufficient for post-translational pre-S translocation. Cell fractionation studies revealed that pre-S refolding and thus the dual topology of L is established at the endoplasmic reticulum (ER) membrane rather than at a post-ER compartment as originally supposed. Together our data provide evidence to suggest that the topological reorientation of L is facilitated by a host cell transmembrane transport machinery such as the ER translocon.

For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)viral particles, regulation of supercoiled DNA amplification, and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we initiated a two-hybrid screen using the L-specific pre-S1 domain as bait. With this approach, we have identified gamma2-adaptin, a putative member of the clathrin adaptor proteins responsible for protein sorting and trafficking, as a specific binding partner of L protein. Evidence for a physical interaction between L protein and gamma2-adaptin was also demonstrated by affinity chromatography and coimmunoprecipitation, and the binding sites were mapped to the L-specific pre-S1 domain and the gamma2-adaptin-specific ear domain. The specificity of the interaction was further sustained by the failure of gamma1-adaptin, a closely related gamma2-adaptin homologue, to associate with L protein. Analysis of an L mutant protein indicates that the L-gamma2-adaptin interaction strictly depends on the pre-S1 domain of transmembrane L protein oriented to the cytosol and thus appears to occur in the cytosolic environment. Interestingly, coexpression of the two interacting partners in transfected cells resulted in recruitment of gamma2-adaptin by L protein onto cis-Golgi-like structures, strongly indicating that the association is physiologically relevant. Together, the results suggest a role for gamma2-adaptin in L-mediated processes of viral biogenesis and/or pathogenesis, such as facilitating and guiding HBV assembly.

Human interleukin-6 has been shown to promote hepatitis B virus (HBV) infection. However, it is not clear whether this influence is the result of a direct interaction between interleukin-6 (IL-6) and the HBV envelope proteins or of a rather indirect mechanism. A direct interaction of IL-6 and the preS region of the large envelope protein (L-protein) of HBV has been reported. In this study we assessed the binding of IL-6 and of the IL-6 receptor subunits to the preS region of the L-protein of HBV. Binding of IL-6 and IL-6 receptor subunits sIL-6R and gp130 to preS was assessed by immunoprecipitation with recombinant preS proteins. In patient sera IL-6 and sIL-6R concentrations were analysed with respect to the course of hepatitis B infection during and after interferon-alpha (IFN-alpha) therapy. The IL-6 and IL-6 receptor subunits could not be precipitated with recombinant preS proteins. In sera of patients who responded to IFN-alpha therapy by virus elimination, a significant increase in sIL-6R concentration was measured. No increase in sIL-6R levels was seen in patients who did not respond to IFN-alpha. Hence, IL-6 and IL-6 receptor subunits do not bind to preS directly. A possible role for sIL-6R in the elimination of HBV infection is discussed.

Among the three related L, M, and S envelope proteins of the hepatitis B virus (HBV), the L and S polypeptides are required for virion production. Whereas the pivotal function of the pre-S region of L in nucleocapsid envelopment has been established, the contribution of its S domain and the S protein is less clear. In this study, we evaluated the role of the cytosolic S loop, common to L and S, in HBV assembly by performing mutagenesis experiments. To distinguish between the effect of the mutations on either envelope or virion formation, we investigated the ability of the mutants to assemble into secretable subviral empty envelopes and to replace the wild-type proteins in virion maturation, respectively. Virion production was found to be blocked by each of the secretion-competent deletion and substitution mutants SDelta35-39, SDelta40-46, SDelta50-56, and Svarsigma56-59, while an insertion within the loop is tolerated. Surprisingly, single mutations of the arginines terminating the loop had an opposite effect: while a conservative exchange of Arg-73 still allowed virion formation, the same mutation of Arg-79 did not. The critical sequences and/or structural requirements of the cytosolic S loop involved in nucleocapsid envelopment primarily act in the S background. These findings can be related to a model for a synergistical function of both L and S proteins in HBV morphogenesis., (Copyright 2000 Academic Press.)

Little is known about host cell factors necessary for hepatitis B virus (HBV) assembly which involves envelopment of cytosolic nucleocapsids by the S, M and L transmembrane viral envelope proteins and subsequent budding into intraluminal cisternae. Central to virogenesis is the L protein that mediates hepatocyte receptor binding and envelopment of capsids. To serve these topologically conflicting roles, L protein exhibits an unusual dual membrane topology, disposing its N-terminal preS domain inside and outside of the virion lipid envelope. The mixed topology is achieved by posttranslational preS translocation of about half of the L protein molecules across a post-endoplasmic reticulum membrane. Here we identify and characterize a preS-specific sequence that confers the suppression of cotranslational translocation even of a model reporter. This cytosolic anchorage sequence specifically binds the cognate heat shock protein Hsc70, thus indicating chaperone participitation in HBV morphogenesis. Conversely, the M envelope protein needs the assistance of the chaperone calnexin for proper folding and trafficking. Calnexin selectively binds to the N-glycan, specific for M, rather than to the N-glycan, common to all three envelope proteins. As inhibition of the calnexin-M interaction blocks the secretion of viral envelopes, we propose an essential role for calnexin, as well as for Hsc70, in chaperoning HBV assembly.

In order to design optimized DNA vectors as genetic vaccines against infections with the hepatitis B virus (HBV) we investigated if secretion or retention of the viral antigens has an influence on the quality and quantity of the humoral immune response. Intramuscular injection of plasmid DNA encoding the HBV large L envelope protein, known to be retained within host cells, induced only a weak response in mice whereas a vector expressing the secretion-competent small S envelope protein elicited strong and sustained immunity. Immunization with rearranged envelope genes further demonstrated that secretion affects the magnitude of the immune response. In situ expression of modified small and middle envelope genes carrying C-terminally attached epitopes are derived from the preS1 region of L generated high titers of preS1- and preS2-specific antibodies, unless antigen secretion was blocked. Accessibility of preS antigens to B-cells that can be achieved by generating extracellular forms of the envelope proteins is thus critical to elicit humoral responses. Such DNA constructs carrying preS1 determinants are promising candidates for the development of multivalent HBV vaccines.

Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly of HBV. Beyond being modified by N glycosylation, M is modified by O glycosylation occurring within its amino acid sequence at positions 27 to 47. The O glycans, however, were found to be dispensable for secretion of M but may rather support viral infectivity. Surprisingly, nonglycosylated M localizes exclusively to the cytosol, either for degradation or for a yet-unknown function.

The large L envelope protein of the hepatitis B virus has the peculiar capacity to adopt two transmembrane topologies. The N-terminal preS domain of L initially remains in the cytosol while the S domain is cotranslationally inserted into the endoplasmic reticulum membrane. The preS region of about half of the L molecules' is posttranslationally translocated to the lumenal space. We now demonstrate that the repression of cotranslational translocation of preS is conferred by a preS1-specific sequence. By analysis of L deletion mutants, the cytosolic anchorage determinant was mapped to amino acid sequence 70 to 94 of L. The intrinsic potential of this determinant to suppress cotranslational translocation was confirmed by transfer to the HBV middle envelope protein. In searching for cellular factors potentially involved in this novel process, we identified the cytosolic heat shock protein Hsc70 as a specific binding partner of L. The interaction site(s) for the chaperone was mapped to amino acids 63 to 107 of L using coimmunoprecipitation and in vitro binding analyses. Deletion of the cytosolic anchorage determinant almost completely abolished ATP-dependent Hsc70 binding. Therefore, interaction between Hsc70 and L is likely to be responsible for the suppression of cotranslational translocation of the preS domain.

The current hepatitis B virus (HBV) vaccines contain the small (S) and middle (M) viral envelope proteins in particulate form but lack the large (L) protein. Although these particles elicit protective immunity to HBV, inclusion of the immunogenic preS1 region of the L protein may enhance their efficacy. To present preS1-derived epitopes on secretable subviral particles we rearranged the HBV envelope ORF by fusing part or all of the preS1 region to either the N or C terminus of the S protein. Fusion of the first 42 residues of preS1 to either site allowed efficient secretion of the modified particles and rendered the linked sequence accessible at the surface of the particle. Conversely, fusion of preS1 sequences to the C terminus of the M protein completely blocked secretion. This block to secretion could be rescued by provision of a heterologous N-terminal signal sequence. All these particles displayed preS1, preS2 and S protein antigenicity. In mice, each construct elicited high titres of preS1-specific antibodies which recognized the authentic L protein. Particles composed of the modified M protein also induced a preS2-specific response. Unexpectedly, however, neither particle elicited S protein-specific antibodies. Nonetheless, the genetic approach employed here represents a strategy to incorporate preS1-derived epitopes both in high density and in highly immunogenic form into their authentic carrier matrix.

The small (S), middle (M) and large (L) envelope proteins of the hepatitis B virus (HBV) are initially synthesized as multispanning membrane proteins of the endoplasmic reticulum membrane. We now demonstrate that all envelope proteins synthesized in transfected cells or in a cell-free system adopt more than one transmembrane orientation. The L protein disposes its N-terminal preS domain both to the cytoplasmic and the luminal side of the membrane. This unusual topology does not depend on interaction with the viral nucleocapsid, but is preserved in secreted empty envelope particles. Pulse-chase analysis suggests a novel process of post-translational translocation leading to the non-uniform topology. Analysis of L deletion mutants indicates that the block to co-translational translocation can be attributed to a specific sequence within preS, suggesting that translocation of L may be regulated. Additional topological heterogeneity is displayed in the S region of the envelope proteins and in the S protein itself, as assayed in a cell-free system. S proteins integrated into microsomal membranes exhibit both a luminal and a cytoplasmic orientation of the internal hydrophilic region carrying the major antigenic determinants. This may explain the unusual partial glycosylation of the HBV envelope proteins.

The 20-nm noninfectious empty envelope particles carrying the hepatitis B surface antigen are secreted in large excess from hepatocytes during a hepatitis B infection. Similar particles produced in cell lines or yeast are an efficient vaccine against hepatitis B virus. We have analyzed the assembly of 20-nm particles using a mutagenesis approach. Specific mutations were introduced into the S gene and the preS region encoding the viral envelope proteins using recombinant DNA techniques. The mutant genes were expressed in cell lines to identify the amino acid residues that are critical for the assembly and the secretion of the 20-nm particles.

The small envelope S protein of hepatitis B virus carrying the surface antigen has the unique property of mobilizing cellular lipids into empty envelope particles which are secreted from mammalian cells. We studied the biogenesis of such particles using site-directed mutagenesis. In this study, we describe the effect of deletions in the N-terminal hydrophobic and hydrophilic domains of the S protein. Whereas short overlapping deletions of hydrophilic sequences flanking the first hydrophobic domain were tolerated, larger deletions of the same sequences were not. Conversely, the hydrophilic region preceding the second hydrophobic domain was not permissive for even short deletions. Deletion of part or all of the first hydrophobic domain also completely blocked secretion, confirming that the entire apolar region serves an essential function. Most of the secretion-defective deletion mutants still entered the secretory pathway and translocated at least the second hydrophilic domain across the membrane of the endoplasmic reticulum. These mutants appeared to remain arrested in a membrane-associated configuration in the endoplasmic reticulum or the cis-Golgi compartment but preserved their capacity for oligomerization with the wild-type S protein. While secretion of wild-type S protein was specifically blocked by the formation of intracellularly retained mixed envelope aggregates, secretion of an unrelated protein (interleukin 9) was completely unaffected.

We have found previously that hybrid 22-nm HBsAg particles can be created by insertion of short antigenic sequences into the HBV major envelope protein. We have now performed a detailed deletion mutagenesis of the S gene of HBV encoding HBsAg. Deletion of the 51 C-terminal amino acids including most of the third and all of the fourth hydrophobic domain of the S protein did not affect particle assembly and secretion. However, secretion of 22-nm particles was abolished by minor deletions in the N-terminal region. Insertion and deletion/substitution mutants carrying a poliovirus epitope at the N-terminus and the preS1 region at the C-terminus have been characterized.

The envelope of hepatitis B virus contains three related proteins, one of which is myristylated. The nonmyristylated small and middle protein are assembled into empty envelope particles which are secreted from cells, whereas the myristylated large envelope protein is mainly found in complete virions and is not secreted in the absence of the nucleocapsid. The block to secretion can be partially overcome by mutation or deletion of the myristylation site. Creation of a myristyl attachment site in the small protein impairs the secretion of empty envelope particles but not their intracellular assembly. Myristylation may therefore play a crucial role in hepatitis B virus replication by channeling the envelope proteins into complete viral particles.

Processes of growth and differentiation of the neurons are based on adequate metabolic activities of the karyoplasm and cytoplasm and measurable at the volume of the cell nucleus. Furthermore, they are affected by metabolic activities related to special neuronal functions. Studies of neurons related to the Nuclei arcuatus, amygdaloideus medialis, amygdaloideus corticalis and amygdaloideus basolateralis (Pars lateralis of the Nucleus amygdaloideus basalis) as well as to the external granular layer (Lamina granularis externa, Lamina II) of the parietal cortex of female and male Wistar rats resulted in distinct karyovolumetrically verifiable variations of the neuronal activities during the postnatal development (between the 5th and 60th day after birth). The variations of the neuronal activities are characterized by their nuclear specifity and indicate a significant sexual dimorphism, which also is of nuclear specifity. The nuclear specifity of the neuronal activity and the variations of the activities are understood as a total of differentiated developmental rhythms and performances of the studied neurons during the postnatal development. The sexual dimorphism, reflected in the nuclear cell volume resp. in the neuronal activity, is finally related to the sexual specificity of the differentiation of the central nervous system.

The Saccharomyces cerevisiae YPT1 gene codes for a ras-like, guanine nucleotide-binding protein which is essential for cell viability. The functional significance of two consecutive cysteines at the very carboxyl-terminal end of this protein and in ypt homologues of other eukaryotic species was examined. YPT1 gene mutations were generated that either led to substitutions by serine or the deletion of one or both C-terminal cysteines. The consequences of the mutations were checked in cells after replacing the wild type with the mutant genes. It was found that as long as one of the cysteines was retained, the protein was fully functional. The YPT1 protein could be labelled with [3H]palmitic acid that appeared to be bound in an ester linkage. The wild-type protein was evenly distributed between soluble and membrane-associated proteins, the palmitoylated form was predominantly in the crude membrane fraction. The mutant protein lacking the C-terminal cysteines was not palmitoylated and was exclusively found in the soluble fraction. The extension by three residues, -Val-Leu-Ser, generating a ras-typical C-terminal end, did not interfere with the mutant YPT1 protein's function although it resulted in a reduced labelling with palmitic acid.

The protein-coding region of the essential Saccharomyces cerevisiae YPT1 gene coding for a ras-related, guanine-nucleotide-binding protein was exchanged in chromosome VI by the protein-coding segment of either the mouse ypt1 gene or the v-Ki-ras gene, and different chimeric YPT1-v-Ki-ras genes. The mouse ypt1 protein with 71% of identical residues compared with the yeast Ypt1 protein could functionally fully replace its yeast homologue as long as the mouse gene was overexpressed under transcriptional control of the inducible GAL10 promoter. In contrast, neither the viral Ki-ras nor the hybrid proteins were able to substitute for the loss of YPT1 gene function. This study suggests that different parts of the yeast Ypt1 protein are required for the interaction with cellular targets and that these essential parts are conserved in the mammalian ypt1 protein.