Elf1p, a Member of the ABC Class of ATPases, Functions as a mRNA Export Factor in Schizosacchromyces pombe

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The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E⅐S, P-glycoprotein⅐ATP) and enzyme-product (E⅐P, P-glycoprotein⅐ ADP⅐P i ) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[␣-32 P]ATP as well as under equilibrium conditions using [␣-32 P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[␣-32 P]ATP or [␣-32 P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E⅐S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E⅐P state continues to have low affinity for substrate, suggesting that conformational changes that form the E⅐S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E⅐S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

Section: Photocross-linking Of 8-azido[␣-32 P]atp Tomentioning

confidence: 99%

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

Sauna

Nandigama

Ambudkar

2006

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“…A pair of primers flanking the ends of the coding sequence of the intron-carrying ␤-tubulin gene was used for RT-PCR to amplify cDNA from its transcripts (29). RNA extracted from ⌬uap56 cells expressing Uap56p produced a single RT-PCR product of ϳ1400 bp, consistent with a fully spliced mRNA (Fig.…”

Section: S Pombe Uap56pmentioning

confidence: 99%

The Nuclear Export Signal of Splicing Factor Uap56p Interacts with Nuclear Pore-associated Protein Rae1p for mRNA Export in Schizosaccharomyces pombe

Thakurta¹,

Selvanathan²,

Patterson

et al. 2007

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Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNAbinding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.Mammalian UAP56 (Saccharomyces cerevisiae Sub2p) and its functional homologs belong to the conserved DECD box class of ATP-dependent RNA helicases that play important functional roles in multiple aspects of DNA and RNA metabolism (1). They are essential in a number of organisms, including yeast, nematode, and fruit fly (2). UAP56/Sub2p is directly involved in pre-messenger RNA splicing and nuclear export of messenger RNAs in S. cerevisiae and in metazoan cells (3). Recently, in Schizosaccharomyces pombe, we found that uap56 is an essential gene for growth and that Uap56p is critical for mRNA export (4). Mammalian UAP56 functions in both ATP-independent and ATP-dependent steps of the spliceosome assembly process (5). Uap56p homologs typically contain a characteristic ATP-binding site, a catalytic DECD box, and an RNA interaction motif. Mutations within the ATP-binding pocket or the catalytic DECD box residues abolished the splicing functions of UAP56 (5).

“…eEF3 stimulates binding of the eEF1⅐GTP⅐aminoacyl-tRNA ternary complex to the ribosomal A site by facilitating the release of the deacylated tRNA from the E site, thus stimulating protein synthesis (8,9). On the other hand, Class IVB contains proteins thought to be important for the export of mRNAs from the nucleus in yeast (10).…”

mentioning

confidence: 99%

ABC50 Promotes Translation Initiation in Mammalian Cells

Paytubi

Wang

Lam

et al. 2009

120

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.