Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.     

Ty3 nucleocapsid controls localization of particle assembly

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX₂CX₄HX₄C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.     

A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein.

Cleavage of the Gag and Gag-Pol polyprotein precursors is a critical step in proliferation of retroviruses and retroelements. The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag protein. Ty1 Gag is not obviously homologous to the Gag proteins of retroviruses. The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation. Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus. A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag. Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs. While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1. Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.     

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.     

Structure of the most conserved internal loop in SRP RNA.

The signal recognition particle (SRP) directs translating ribosomes to the protein translocation apparatus of endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. The SRP is universally conserved, and in prokaryotes consists of two essential subunits, SRP RNA and SRP54, the latter of which binds to signal sequences on the nascent protein chains. Here we describe the solution NMR structure of a 28-mer RNA composing the most conserved part of SRP RNA to which SRP54 binds. Central to this function is a six-nucleotide internal loop that assumes a novel Mg2+-dependent structure with unusual cross-strand interactions; besides a cross-strand A/A stack, two guanines form hydrogen bonds with opposite-strand phosphates. The structure completely explains the phylogenetic conservation of the loop bases, underlining its importance for SRP54 binding and SRP function.     

A complex of the signal sequence binding protein and the SRP RNA promotes translocation of nascent proteins.

Translocation of proteins across the endoplasmic reticulum membrane is initiated by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex consisting of a 7S RNA and six polypeptides. To investigate the functions of the SRP components, we have tested the activities of several SRP subparticles. We show that the SRP GTPase (SRP54) alone binds a signal sequence and discriminates it from a non-signal sequence. Although SRP54 alone is unable to promote translocation, SRP54 in a complex with SRP RNA is both necessary and sufficient to promote translocation of an elongation-arrested nascent protein in a GTP-regulated manner. For co-translational translocation, additional SRP components are required. We discuss the implications of our results for the function of the Escherichia coli SRP which is homologous to the SRP54/SRP-RNA complex.     

Ty1 Gag Enhances the Stability and Nuclear Export of Ty1 mRNA

Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus‐like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1‐less strain expressing galactose‐inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense‐mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.     

The TY3 Gag3 spacer controls intracellular condensation and uncoating

Cells expressing the yeast retrotransposon Ty3 form concentrated foci of Ty3 proteins and RNA within which virus-like particle (VLP) assembly occurs. Gag3, the major structural protein of the Ty3 retrotransposon, is composed of capsid (CA), spacer (SP), and nucleocapsid (NC) domains analogous to retroviral domains. Unlike the known SP domains of retroviruses, Ty3 SP is highly acidic. The current studies investigated the role of this domain. Although deletion of Ty3 SP dramatically reduced retrotransposition, significant Gag3 processing and cDNA synthesis occurred. Mutations that interfered with cleavage at the SP-NC junction disrupted CA-SP processing, cDNA synthesis, and electron-dense core formation. Mutations that interfered with cleavage of CA-SP allowed cleavage of the SP-NC junction, production of electron-dense cores, and cDNA synthesis but blocked retrotransposition. A mutant in which acidic residues of SP were replaced with alanine failed to form both Gag3 foci and VLPs. We propose a speculative "spring" model for Gag3 during assembly. In the first phase during concentration of Gag3 into foci, intramolecular interactions between negatively charged SP and positively charged NC domains of Gag3 limit multimerization. In the second phase, the NC domain binds RNA, and the bound form is stabilized by intermolecular interactions with the SP domain. These interactions promote CA domain multimerization. In the third phase, a negatively charged SP domain destabilizes the remaining CA-SP shell for cDNA release.     

Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry

Fluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition.     

NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.     

ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.     

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.     

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.     

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.     

Unraveling the components of protein translocation pathway in human malaria parasite Plasmodium falciparum

The targeting and translocation of proteins is an essentially required and conserved process in all the living organisms. This complex process involves multiple steps and requires a variety of factors before the protein reaches its final destination. The major components of translocation machinery are signal recognition particle (SRP) and secretory (Sec) complex. These are composed of highly conserved components. SRP contains SRP RNA and other polypeptides such as SRP9, SRP14, SRP19 and SRP54. Sec complex is composed of Sec61αβγ, Sec62 and Sec63. In this review using bioinformatics approach we have shown that the P. falciparum genome contains the homologues for all of these and other factors such as SRP receptor, and TRAM (translocation associated membrane protein), which are required for post- and co-translational protein translocation. We have also shown the various steps of translocation in a hypothetical model.     

Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor.

The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively. The ability of these E. coli components to function in a bona fide co-translational targeting pathway remains unclear. Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro. Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally. Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery. The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane. Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.     

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences.

The signal recognition particle (SRP) binds to signal sequences when they emerge from a translating ribosome and targets the complex of ribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co-translational translocation of the nascent chain. By photo-crosslinking it has been shown that the signal sequence of preprolactin (PPL) only interacts with the methionine-rich (M) domain of the 54 kDa protein subunit (SRP54) of SRP. Here we show that (i) a signal-anchor sequence is likewise crosslinked only to the methionine-rich domain of SRP54, (ii) free SRP54 can interact with signal sequences independently of the other components of SRP, (iii) its M domain suffices to perform this function, and (iv) an essentially intact M domain is required for signal sequence recognition. Alkylation of the N+G domain in intact SRP54 with N-ethyl maleimide (NEM), but not after cleavage with V8 protease, prevents the binding of a signal sequence to the M domain. This suggests a proximity between the N+G and M domains of SRP54 and raises the possibility that the role of the N+G domain may be to regulate the binding and/or the release of signal sequences.     

Transport of the intracisternal A-type particle Gag polyprotein to the endoplasmic reticulum is mediated by the signal recognition particle

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.