scholarly journals Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

2010 ◽  
Vol 84 (18) ◽  
pp. 9035-9046 ◽  
Author(s):  
Mirela Popa ◽  
Zsolt Ruzsics ◽  
Mark Lötzerich ◽  
Lars Dölken ◽  
Christopher Buser ◽  
...  
Keyword(s):  
Unit Length ◽  
Virus Production ◽  
Dominant Negative ◽  
Murine Cytomegalovirus ◽  
Phenotypic Characterization ◽  
Reading Frame ◽  
Viral Capsids ◽  
Gene Block ◽  
Nuclear Egress ◽  
C Terminus

ABSTRACT The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.

scholarly journals Functional Domains of Murine Cytomegalovirus Nuclear Egress Protein M53/p38

2006 ◽  
Vol 80 (1) ◽  
pp. 73-84 ◽  
Author(s):  
Mark Lötzerich ◽  
Zsolt Ruzsics ◽  
Ulrich H. Koszinowski
Keyword(s):  
Binding Site ◽  
Random Mutagenesis ◽  
Murine Cytomegalovirus ◽  
Homologous Proteins ◽  
Herpes Simplex Virus 1 ◽  
Nuclear Egress ◽  
C Terminus ◽  
Localization Signal ◽  
Definition Of ◽  
Simplex Virus

ABSTRACT Two conserved herpes simplex virus 1 proteins, UL31 and UL34, form a complex at the inner nuclear membrane which governs primary envelopment and nuclear egress of the herpesvirus nucleocapsids. In mouse cytomegalovirus, a member of the betaherpesvirus subfamily, the homologous proteins M53/p38 and M50/p35 form the nuclear egress complex (NEC). Since the interaction of these proteins is essential for functionality, the definition of the mutual binding sites is a prerequisite for further analysis. Using a comprehensive random mutagenesis procedure, we have mapped the M53/p38 binding site of M50/p35 (A. Bubeck, M. Wagner, Z. Ruzsics, M. Lötzerich, M. Iglesias, I. R. Singh, and U. H. Koszinowski, J. Virol. 78:8026-8035). Here we describe a corresponding analysis for the UL31 homolog M53/p38. A total of 72 individual mutants were reinserted into the genome to test the complementation of the lethal M53 null phenotype. The mutants were also studied for colocalization and for coprecipitation with M50/p35. The analysis revealed that the nonconserved N-terminal one-third of M53/p38 provides the nuclear localization signal as an essential function. The collective results for many mutants localized the binding site for M50/p35 to amino acids (aa) 112 to 137. No single aa exchange for alanine could destroy NEC formation, but virus attenuation revealed a major role for aa K128, Y129, and L130. The lethal phenotype of several insertion and stop mutants indicated the functional importance of the C terminus of the protein.

scholarly journals The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

2007 ◽  
Vol 82 (5) ◽  
pp. 2065-2078 ◽  
Author(s):  
Eva Maria Borst ◽  
Karen Wagner ◽  
Anne Binz ◽  
Beate Sodeik ◽  
Martin Messerle
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Essential Role ◽  
Unit Length ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Viral Genomes ◽  
Nuclear Egress ◽  
C Terminus ◽  
Infected Cells

ABSTRACT Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.

scholarly journals Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

2014 ◽  
Vol 89 (4) ◽  
pp. 2002-2012 ◽  
Author(s):  
Lars Paßvogel ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Thomas C. Mettenleiter
Keyword(s):  
Nuclear Export ◽  
Pseudorabies Virus ◽  
Functional Characterization ◽  
Nuclear Export Signal ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Nuclear Targeting ◽  
Nuclear Egress ◽  
C Terminus ◽  
Negative Effect

ABSTRACTThe herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.IMPORTANCEThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

scholarly journals Random Screening for Dominant-Negative Mutants of the Cytomegalovirus Nuclear Egress Protein M50

2007 ◽  
Vol 81 (11) ◽  
pp. 5508-5517 ◽  
Author(s):  
Brigitte Rupp ◽  
Zsolt Ruzsics ◽  
Christopher Buser ◽  
Barbara Adler ◽  
Paul Walther ◽  
...  
Keyword(s):  
Variable Region ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Murine Cytomegalovirus ◽  
Homologous Gene ◽  
Sequence Motif ◽  
Loss Of Function ◽  
Nuclear Egress ◽  
Step Procedure ◽  
Conditional Expression

ABSTRACT Inactivation of gene products by dominant-negative (DN) mutants is a powerful tool to assign functions to proteins. Here, we present a two-step procedure to establish a random screen for DN alleles, using the essential murine cytomegalovirus gene M50 as an example. First, loss-of-function mutants from a linker-scanning library were tested for inhibition of virus reconstitution with the help of FLP-mediated ectopic insertion of the mutants into the viral genome. Second, DN candidates were confirmed by conditional expression of the inhibitory proteins in the virus context. This allowed the quantification of the inhibitory effect, the identification of the morphogenesis block, and the construction of DN mutants with improved activity. Based on these observations a DN mutant of the homologous gene (UL50) in human cytomegalovirus was predicted and constructed. Our data suggest that a proline-rich sequence motif in the variable region of M50/UL50 represents a new functional site which is essential for nuclear egress of cytomegalovirus capsids.

scholarly journals C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Fu-Hsien Yu ◽  
Kuo-Jung Huang ◽  
Chin-Tien Wang
Keyword(s):  
Leucine Zipper ◽  
Virus Assembly ◽  
Virus Production ◽  
Supporting Evidence ◽  
Dependent Manner ◽  
Dimer Formation ◽  
Reading Frame ◽  
Virus Maturation ◽  
C Terminus ◽  
Hiv 1

ABSTRACT HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS.

scholarly journals Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

2014 ◽  
Vol 89 (1) ◽  
pp. 523-534 ◽  
Author(s):  
Mayuri Sharma ◽  
Brian J. Bender ◽  
Jeremy P. Kamil ◽  
Ming F. Lye ◽  
Jean M. Pesola ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Continuous Distribution ◽  
Virus Production ◽  
Nuclear Lamina ◽  
Dominant Negative ◽  
Lamin A ◽  
Nuclear Egress ◽  
Dividing Cells ◽  
Infected Cells

ABSTRACTHerpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with aUL97mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

scholarly journals The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

2016 ◽  
Vol 90 (13) ◽  
pp. 5860-5875 ◽  
Author(s):  
Eva Maria Borst ◽  
Rudolf Bauerfeind ◽  
Anne Binz ◽  
Thomas Min Stephan ◽  
Sebastian Neuber ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Fluorescent Protein ◽  
Unit Length ◽  
Start Codon ◽  
Nuclear Targeting ◽  
Reading Frame ◽  
A Genome ◽  
Genome Encapsidation ◽  
Insight Into

ABSTRACTSeveral essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

scholarly journals A disease-associated frameshift mutation in caveolin-1 disrupts caveolae formation and function through introduction of a de novo ER retention signal

2017 ◽  
Vol 28 (22) ◽  
pp. 3095-3111 ◽  
Author(s):  
Courtney A. Copeland ◽  
Bing Han ◽  
Ajit Tiwari ◽  
Eric D. Austin ◽  
James E. Loyd ◽  
...  
Keyword(s):  
Frameshift Mutation ◽  
De Novo ◽  
Dominant Negative ◽  
Caveolin 1 ◽  
Protein Levels ◽  
C Terminus ◽  
Er Retention ◽  
And Function ◽  
Er Retention Signal ◽  
Retention Signal

Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1–/– MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1–/– MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.

Multimeric connexin interactions prior to the trans-Golgi network

2001 ◽  
Vol 114 (22) ◽  
pp. 4013-4024
Author(s):  
Jayasri Das Sarma ◽  
Rita A. Meyer ◽  
Fushan Wang ◽  
Valsamma Abraham ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Dominant Negative ◽  
Alveolar Epithelial Cells ◽  
Trans Golgi Network ◽  
Alveolar Epithelial ◽  
C Terminus ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Nih 3T3 ◽  
Gap Junction Hemichannels

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial β-galactosidase fused to the C terminus of connexin43 (Cx43/β-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/β-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a subhexameric complex. Cx43/β-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/β-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/β-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

scholarly journals Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo

2004 ◽  
Vol 377 (2) ◽  
pp. 459-467 ◽  
Author(s):  
Jose M. LAPLAZA ◽  
Magnolia BOSTICK ◽  
Derek T. SCHOLES ◽  
M. Joan CURCIO ◽  
Judy CALLIS
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein A ◽  
Fold Increase ◽  
Lysine Residue ◽  
Reading Frame ◽  
E3 Ligases ◽  
C Terminus ◽  
Dependent Modification ◽  
F Box Protein

In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1–Cdc53–F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2/ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1- and ENR2-independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101Δ strain was hypersensitive to thiabendazole, isopropyl (N-3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1Δ strains were not. Whereas rtt101Δ strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1Δ strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1-dependent nor the RUB1-independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.

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