Jan C. Vos

The major substrates for TAP in vivo are derived from newly synthesized proteins.

The transporter associated with antigen processing (TAP) is a member of the family of ABC transporters that translocate a large variety of substrates across membranes. TAP transports peptides from the cytosol into the endoplasmic reticulum for binding to MHC class I molecules and for subsequent presentation to the immune system. Here we follow the lateral mobility of TAP in living cells. TAPs mobility increases when it is inactive and decreases when it translocates peptides. Because TAP activity is dependent on substrate, the mobility of TAP is used to monitor the intracellular peptide content in vivo. Comparison of the diffusion rates in peptide-free and peptide-saturated cells indicates that normally about one-third of all TAP molecules actively translocate peptides. However, during an acute influenza infection TAP becomes fully employed owing to the production and degradation of viral proteins. Furthermore, TAP activity depends on continuing protein translation. This implies that MHC class I molecules mainly sample peptides that originate from newly synthesized proteins, to ensure rapid presentation to the immune system.

TC1 TRANSPOSASE OF CAENORHABDITIS ELEGANS IS AN ENDONUCLEASE WITH A BIPARTITE DNA BINDING DOMAIN

The Tc1 transposon of Caenorhabditis elegans is a member of the Tc1/mariner family of mobile elements. These elements have inverted terminal repeats that flank a single transposase gene. Here we show that Tc1 transposase, Tc1A, has a bipartite DNA binding domain related to the paired domain of mammalian and Drosophila genes. Both the DNA binding domain of Tc1A and the DNA binding site in the inverted repeat of Tc1 can be divided into two subdomains. Methylation interference studies demonstrate adjacent minor and major groove contacts at the inner part of the binding site by the N‐terminal 68 amino acids of the DNA binding domain. In addition, Tc1A amino acids 69‐142 are essential for major groove contacts at the outer part of the binding site. Recombinant Tc1A is found to be able to introduce a single strand nick at the 5′ end of the transposon in vitro. Furthermore, Tc1A can mediate a phosphoryl transfer reaction. A mutation in a DDE motif abolishes both endonucleolytic and phosphoryl transfer activities, suggesting that Tc1A carries a catalytic core common to retroviral integrases and IS transposases.

Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5' poly(A) leader

Abstract We have demonstrated by primer elongation and cap analysis that mature vaccinia virus late transcripts are discontinuously synthesized. We have shown that RNA transcripts from a translocated 11K and from the authentic 11K and 4b late promoters are extended by approximately 35 nucleotides beyond the “start site” determined by S1 mapping using vaccinia genomic DNA as a probe. Sequencing of the RNA and of the first strand cDNA reveal that a homopolymeric poly(A) sequence is linked to the 5′ terminus of the RNA transcripts. S1 mapping of RNA transcripts with a DNA probe containing an A-stretch, replacing promoter sequences upstream of position −1, confirms the existence of a poly(A) leader of approximately 35 A-residues.

Derepression of a novel class of vaccinia virus genes upon DNA replication

A novel class of vaccinia virus genes, called intermediate, is expressed immediately post‐replication and prior to the onset of late gene transcription. Intermediate transcription is dependent on trans‐acting factors which are present in an active state in virus‐infected cells prior to the onset of DNA replication. Plasmid‐borne intermediate genes transfected into vaccinia‐virus infected cells are expressed prior to DNA replication, whereas the copies within the viral genome are repressed. DNA replication is essential for activation of viral intermediate transcription and de novo protein synthesis is not required post‐replication. In contrast, activation of late transcription depends on DNA replication and continued de novo protein synthesis. Therefore, a subset of intermediate proteins is likely to be trans‐activators of late gene transcription. Cell‐free extracts differentially transcribe early, intermediate and late genes in a way similar to the temporal expression observed in vivo. A cascade model is discussed for the regulation of gene expression during the viral life‐cycle.

Vaccinia virus capping enzyme is a transcription initiation factor

It has previously been demonstrated that vaccinia virus capping enzyme is involved both in the formation of a 5′ cap structure and in termination of early transcription. Here we show that capping enzyme has an additional activity which is required for transcription of intermediate genes. VITF‐A and VITF‐B have been defined as two activities which together with RNA polymerase are necessary and sufficient to transcribe intermediate genes in vitro. VITF‐A and the viral capping enzyme are shown to copurify to near homogeneity. Direct evidence that capping enzyme is VITF‐A was obtained by complementation of a reconstituted transcription system with viral capping enzyme expressed in Escherichia coli. Although capping enzyme is a cofactor in early transcription termination, intermediate transcription is not terminated in response to the early termination signal. Capping enzyme is shown to form a complex with RNA polymerase in the absence of VITF‐B. This appears to be a prerequisite for the formation of a stable initiation complex.

Head–head/tail–tail relative orientation of the pore-forming domains of the heterodimeric ABC transporter TAP

BACKGROUND The transporter associated with antigen processing (TAP) is a heterodimeric member of the large family of ABC transporters. The study of interactions between the subunits TAP1 and TAP2 can reveal the relative orientation of the transmembrane segments, which form a translocation pore for peptides. This is essential for understanding the architecture of TAP and other ABC transporters. RESULTS The amino-terminal six transmembrane segments (TMs) of human TAP1, TAP1 (1-6), and the amino-terminal five TMs of TAP2, TAP2(1-5), are thought to constitute the pore of TAP. Two new approaches are used to define dimer interactions. We show that TM6 of TAP1 (1-6) is able to change topology post-translationally. This TM, along with a cytoplasmic tail, is translocated into the endoplasmic reticulum lumen, unless TAP2 is expressed. Coexpression of TM(4-5) of TAP2 stabilizes the topology of TAP1 (1-6), even when the TM1 of TAP1 is subsitituted with another sequence. This suggests that the carboxy-terminal TMs of the pore-forming domains TAP1 (1-6) and TAP2(1-5) interact. An alternative assay uses photobleaching in living cells using TAP1 (1-6) tagged with the green fluorescent protein (GFP). Coexpression with TAP2(1-5) results in reduced movement of the heterodimer within the endoplasmic reticulum membrane, as compared with the single TAP1 (1-6) molecule. In contrast, TAP2(1-4) has no effect on the mobility of TAP1 (1-6)-GFP, indicating the importance of TM5 of TAP2 for dimer formation. Also, TM1 of both TAP1 and TAP2 is essential for formation of a complex with low mobility. CONCLUSIONS Dimerization of the pore-forming transmembrane domains of TAP1 (TM1-6) with its TAP2 counterpart (TM1-5) prevents the post-translational translocation of TM6 of TAP1 and results in a complex with reduced mobility within the endoplasmic reticulum membrane compared with the free subunit. These techniques are used to show that the pore-forming domains of TAP are aligned in a head-head/tail-tail orientation. This positions the following peptide-binding segments of the two TAP subunits to one side of the pore.

Promoter melting by a stage-specific vaccinia virus transcription factor is independent of the presence of RNA polymerase

Fractionation of an extract prepared from HeLa cells infected with vaccinia virus resulted in the separation of factors involved in vaccinia virus intermediate transcription. Two activities, VITF-A and VITF-B, in addition to the viral RNA polymerase are necessary and sufficient to direct intermediate transcription in vitro. VITF-B confers intermediate promoter specificity to an early-specific extract prepared from virus particles. A committed complex between VITF-B and the template can sequester VITF-A and RNA polymerase into a pre-initiation complex. VITF-B is further able to melt the promoter at the start site of transcription. Open complex formation is stimulated by ATP. In contrast to prokaryotic and eukaryotic pol III transcription, promoter melting is independent of the presence of RNA polymerase.

Deletion of the three distal S1 motifs of Saccharomyces cerevisiae Rrp5p abolishes pre-rRNA processing at site A(2) without reducing the production of functional 40S subunits

ABSTRACT Yeast Rrp5p, one of the few trans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A0 to A2 and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A3 and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutant rrp5Δ6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in the rrp5Δ6 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A2. Instead, the 32S precursor is processed directly at site A3, producing exclusively 21S rather than 20S pre-rRNA. This is the first evidence that the 21S precursor, which was observed previously only in cells showing a substantial growth defect or as a minor species in addition to the normal 20S precursor, is an efficient substrate for 18S rRNA synthesis. Maturation of the 21S precursor occurs via the same endonucleolytic cleavage at site D as that used for 20S pre-rRNA maturation. The resulting D-A3 fragment, however, is degraded by both 5′→3′ and 3′→5′ exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 5′→3′ exonucleolytic digestion of the D-A2 fragment. We also show that rrp5Δ6 cells are hypersensitive to both hygromycin B and cycloheximide, suggesting that, despite their wild-type growth rate, their preribosomes or ribosomes may be structurally abnormal.

Subunits Rip1p and Cox9p of the respiratory chain contribute to diclofenac-induced mitochondrial dysfunction.

The widely used drug diclofenac can cause serious heart, liver and kidney injury, which may be related to its ability to cause mitochondrial dysfunction. Using Saccharomyces cerevisiae as a model system, we studied the mechanisms of diclofenac toxicity and the role of mitochondria therein. We found that diclofenac reduced cell growth and viability and increased levels of reactive oxygen species (ROS). Strains increasingly relying on respiration for their energy production showed enhanced sensitivity to diclofenac. Furthermore, oxygen consumption was inhibited by diclofenac, suggesting that the drug inhibits respiration. To identify the site of respiratory inhibition, we investigated the effects of deletion of respiratory chain subunits on diclofenac toxicity. Whereas deletion of most subunits had no effect, loss of either Rip1p of complex III or Cox9p of complex IV resulted in enhanced resistance to diclofenac. In these deletion strains, diclofenac did not increase ROS formation as severely as in the wild-type. Our data are consistent with a mechanism of toxicity in which diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death.

In vitro recognition of an orf virus early promoter in a vaccinia virus extract.

SummaryDNA fragments containing varying lengths of the 5′ end of an orf virus early gene (ORF 3) and its associated promoter were introduced into sodium deoxycholate-solubilized vaccinia virus extracts capable of initiating transcription in vitro from vaccinia virus early promoters. After separation of the radiolabelled products of the reactions on a 5% polyacrylamide/7 M urea gel, discrete transcripts were detected the sizes of which were consistent with initiation of transcription from the orf virus early promoter. This is the first demonstration in a functional assay of the conservation of early transcriptional promoters between an orthopoxvirus and a parapoxvirus.