Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays

Wg, Dougherty; Td, Parks

doi:10.1016/0042-6822(89)90116-5

Cited by 67 publications

(31 citation statements)

References 28 publications

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“…Biotin-protein ligase covalently couples biotin to residue Lys 12 . Residue Glu 17 functions as the C-terminal residue of the biotin ligase consensus and the N-terminal glutamate of the TEV protease consensus site (ENLYFQG) (35,36). Cloning into the BamHI site leaves a single serine between the C terminus of the TEV consensus site and the sequence of interest.…”

Section: Methodsmentioning

confidence: 99%

Thermodynamics of β-Catenin-Ligand Interactions

Choi

Huber²,

Weis

2006

Journal of Biological Chemistry

144

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␤-Catenin is a structural component of adherens junctions, where it binds to the cytoplasmic domain of cadherin cell adhesion molecules. ␤-Catenin is also a transcriptional coactivator in the Wnt signaling pathway, where it binds to Tcf/Lef family transcription factors. In the absence of a Wnt signal, nonjunctional ␤-catenin is present in a multiprotein complex containing the proteins axin and adenomatous polyposis coli (APC), both of which bind directly to ␤-catenin. The thermodynamics of ␤-catenin binding to E-cadherin, Lef-1, APC, axin, and the transcriptional inhibitor ICAT have been determined by isothermal titration calorimetry. Most of the interactions showed large, unfavorable entropy changes, consistent with these ligands being natively unstructured in the absence of ␤-catenin. Phosphorylation of serine residues present in a sequence motif common to cadherins and APC increased the affinity for ␤-catenin 300 -700-fold, and surface plasmon resonance measurements revealed that phosphorylation of E-cadherin both enhanced its on rate and decreased its off rate. The effects of the N-and C-terminal "tails" that flank the ␤-catenin armadillo repeat domain on ligand binding have also been investigated using constructs lacking one or both tails. Contrary to earlier studies that employed less direct binding assays, the tails did not affect the affinity of ␤-catenin for tight ligands such as E-cadherin, Lef-1, and phosphorylated APC. However, the ␤-catenin C-terminal tail was found to decrease the affinity for the weaker ligands APC and axin, suggesting that this region may have a regulatory role in ␤-catenin degradation.The protein ␤-catenin serves several roles in the development and maintenance of multicellular organisms. It is a structural component of cell-cell contacts and is also a transcriptional coactivator in the Wnt signaling pathway that controls cell fate determination. The multiple functions of this protein depend upon its interactions with several distinct protein ligands. In adherens junctions, ␤-catenin interacts with the cytoplasmic domain of classical cadherins and with ␣-catenin, which functionally connects the cadherin-catenin complex to the actin cytoskeleton. The levels of nonjunctional ␤-catenin are determined by the presence or absence of Wnt growth factor stimulation. In the absence of a Wnt, cytosolic ␤-catenin is targeted for degradation by a multiprotein complex containing axin and the adenomatous polyposis coli protein (APC).3 The axin-APC complex binds to ␤-catenin and also recruits casein kinase 1 (CK1) and glycogen synthase kinase-3␤ (GSK-3␤). These kinases phosphorylate ␤-catenin, flagging it for destruction by the ubiquitin-proteosome pathway. In the presence of a Wnt signal, phosphorylation of ␤-catenin is inhibited. The resulting stabilized pool of ␤-catenin translocates to the nucleus, where it binds to the Tcf/Lef family transcription factors. The ␤-catenin-Tcf complex recruits general transcription factors, resulting in a complex that activates Wnt target genes. The primary s...

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Section: Methodsmentioning

confidence: 99%

Thermodynamics of β-Catenin-Ligand Interactions

Choi

Huber²,

Weis

2006

Journal of Biological Chemistry

144

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“…In the current study, we have expressed the tobacco etch virus (TEV) protease (15,16) in the cytosol or in mitochondria of yeast and asked whether fumarase harboring the protease recognition site can be cleaved by the cytosolic protease during import in vivo. Additionally, the mitochondrially targeted TEV protease enables us to establish an import kinetics assay to determine in vivo the translation coupled import time frame.…”

mentioning

confidence: 99%

Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Yogev

Karniely

Pines

2007

Journal of Biological Chemistry

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Fumarase represents proteins that cannot be imported into mitochondria after the termination of translation (post-translationally). Utilizing mitochondrial and cytosolic versions of the tobacco etch virus (TEV) protease, we show that mitochondrially targeted fumarase harboring a TEV protease recognition sequence is efficiently cleaved by the mitochondrial but not by the cytosolic TEV protease. Nonetheless, fumarase was readily cleaved by cytosolic TEV when its import into mitochondria was slowed down by either (i) disrupting the activity of the TOM complex, (ii) lowering the growth temperature, or (iii) reducing the inner membrane electrochemical potential. Accessibility of the fumarase nascent chain to TEV protease under such conditions was prevented by low cycloheximide concentrations, which impede translation. In addition, depletion of the ribosome-associated nascent polypeptide-associated complex (NAC) reduced the fumarase rate of translocation into mitochondria and exposed it to TEV cleavage in the cytosol. These results indicate that cytosolic exposure of the fumarase nascent chain depends on both translocation and translation rates, allowing us to discuss the possibility that import of fumarase into mitochondria occurs while the ribosome is still attached to the nascent chain.

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“…Fig. 6(a) shows the positions in the BaYMV 270K protein of the three types of dipeptide (QA, QG and QS) which are known to be cleavage sites for the NIa proteinase (Dougherty et al, 1989 a). Comparisons of the sequences surrounding these dipeptides with the sequence found at the verified junction between the putative polymerase and capsid protein of BaYMV (site E) provide the four other possible cleavage sites shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Detailed analyses of the cleavage specificities of the potyvirus NIa proteinase have revealed a clear consensus for the sequence flanking the cleavage sites Dougherty & Parks, 1989;Dougherty et al, 1989a). Fig.…”

Section: Discussionmentioning

confidence: 99%

Nucleotide Sequence of Barley Yellow Mosaic Virus RNA 1: A Close Evolutionary Relationship with Potyviruses

Kashiwazaki

Minobe²,

Omura³

et al. 1990

Journal of General Virology

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The complete nucleotide sequence of barley yellow mosaic virus (BaYMV) RNA 1 was obtained by analysis of overlapping cDNA clones and by direct RNA sequencing. The sequence is 7632 nucleotides in length, excluding a 3' poly(A) tail. The first AUG codon at nucleotide 172 appeared to be the initiator for a single long open reading frame encoding a protein of 2410 amino acids with an Mr of 270755. Amino acid sequence comparisons revealed that the BaYMV 270K protein contains three regions upstream of the Cterminal capsid protein which share significant homologies with the cytoplasmic inclusion and two nuclear inclusion proteins of potyviruses thus indicating their similarities in genetic organization. However, the apparent low levels of homology in the corresponding proteins of BaYMV and potyviruses are in contrast with the high conservation among potyviruses. Moreover, our data indicate that BaYMV RNA 1 has no counterpart to the two cistrons located in the 5'-terminal region of the potyvirus genome. Although the data suggest a close evolutionary relationship between BaYMV and potyviruses, the striking differences set BaYMV apart from potyviruses.

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Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays

Cited by 67 publications

References 28 publications

Thermodynamics of β-Catenin-Ligand Interactions

Thermodynamics of β-Catenin-Ligand Interactions

Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Nucleotide Sequence of Barley Yellow Mosaic Virus RNA 1: A Close Evolutionary Relationship with Potyviruses

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