Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1.

E1 is the largest open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) and is highly conserved among all papillomaviruses, maintaining its size, amino acid composition, and location in the viral genome with respect to other early genes. Multiple viral replication functions have been mapped to the E1 ORF of BPV-1, and evidence suggested that more than one protein was encoded by this ORF. We previously identified a small protein (M) whose gene consists of two exons, one encoded by the 5' end of the E1 ORF. We show here that a 68-kilodalton (kDa) phosphoprotein made from the E1 ORF can be detected in BPV-1-transformed cells, and we present evidence that this protein is encoded by sequences colinear with the entire E1 ORF. The full-length E1 protein immunoprecipitated from virally transformed cells and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comigrates with a protein expressed from a recombinant DNA construct capable of producing only the complete E1 protein. In addition, two different antisera directed against polypeptides encoded from either the 3' or the 5' end of the E1 ORF both recognize the full-length E1 product. A mutation converting the first methionine codon in the ORF to an isoleucine codon abolishes BPV-1 plasmid replication and E1 protein production. Consistent with the notion that this methionine codon is the start site for E1, a mutant with a termination codon placed after the splice donor at nucleotide 1235 in E1 produces a truncated protein with the molecular mass predicted from the primary sequence as well as the previously identified M protein. When visualized by immunostaining, the E1 protein expressed in COS cells is localized to the cell nucleus. A high degree of similarity exists between the BPV-1 E1 protein and polyomavirus and simian virus 40 large-T antigens in regions of the T antigens that bind ATP. We show by ATP affinity labeling that the E1 protein produced in COS cells binds ATP and that this activity is abolished by a point mutation which converts the codon for proline 434 to serine. Furthermore, this mutation renders the viral genome defective for DNA replication, suggesting that the ATP-binding activity of E1 is necessary for its putative role in viral DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blm10 facilitates nuclear import of proteasome core particles

Short‐lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT‐like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP‐CP assemblies are primarily nuclear and promote cell division. During quiescence, RP‐CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10‐bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG‐rich nucleoporins and is dissociated from the CP by Ran‐GTP. Thus, Blm10 represents the first CP‐dedicated nuclear import receptor in yeast.     

Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.     

Silverleaf whitefly stress impairs sugar export from cotton source leaves

Silverleaf whitefly (SLW), Bemisia argentifolii Bellows and Perring, is one of the most noxious pests of numerous field and vegetable crops, causing billions of dollars worth of damage throughout the world. SLW is a phloem feeder whose feeding is likely to interfere with phloem transport. The aim of this study was to test the hypothesis that SLW infestation impairs carbohydrate export from source leaves, and consequently increases their carbohydrate content. The youngest fully expanded leaves of cotton ( Gossypium hirsutum L., cv. Siv'on), grown under SLW-infested and noninfested conditions, were characterized for their diurnal changes in carbohydrate content and photoassimilate export. SLW infestation induced a considerable reduction in net photosynthetic rate (P_n), coupled with increased sucrose, glucose and fructose and decreased starch concentrations. Export rate was determined after 14 CO₂ pulse-labeling both by in situ monitoring of leaf radioactivity and by analyzing the content and radioactivity of the major carbon metabolites. Radioactive counting indicated a lower rate of 14 C efflux for the infested plants. A similar trend was found for the specific activities of sucrose and the three soluble sugars combined (sucrose, glucose and fructose). A single exponential decay function with asymptote was fitted to the above efflux curves. All the calculated exponential coefficients demonstrated lower export rates after SLW injury. These results indicate that SLW impairs photoassimilate export, suggesting possible down-regulation of P_n due to increased foliar soluble sugar contents.     

The Minimal Deneddylase Core of the COP9 Signalosome Excludes the Csn6 MPN(-) Domain

The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1-8), yet in several Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN(-) domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN(-) domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN(-) domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.