scholarly journals A Cell-Free Translation and Proteoliposome Reconstitution System for Functional Analysis of Plant Solute Transporters

2007 ◽  
Vol 48 (12) ◽  
pp. 1815-1820 ◽  
Author(s):  
Akira Nozawa ◽  
Hideaki Nanamiya ◽  
Takuji Miyata ◽  
Nicole Linka ◽  
Yaeta Endo ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Free Translation ◽  
A Cell ◽  
Solute Transporters

scholarly journals Activation of the Kexin from Schizosaccharomyces pombeRequires Internal Cleavage of Its Initially Cleaved Prosequence

1998 ◽  
Vol 18 (1) ◽  
pp. 400-408 ◽  
Author(s):  
Dale Powner ◽  
John Davey
Keyword(s):  
Cleavage Site ◽  
Secretory Pathway ◽  
Catalytic Domain ◽  
Activation Process ◽  
Processing Enzymes ◽  
Free Translation ◽  
A Cell ◽  
Internal Site ◽  
Primary Cleavage ◽  
Full Activation

ABSTRACT Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeastSchizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopuseggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102↓ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombecells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.

scholarly journals A Maternal Smad Protein Regulates Early Embryonic Apoptosis in Xenopus laevis

2002 ◽  
Vol 22 (5) ◽  
pp. 1317-1328 ◽  
Author(s):  
Yuko Miyanaga ◽  
Ingrid Torregroza ◽  
Todd Evans
Keyword(s):  
Functional Analysis ◽  
Cell Survival ◽  
Caspase 3 ◽  
Embryonic Cell ◽  
Early Embryogenesis ◽  
Cell Cycles ◽  
Smad Proteins ◽  
Smad Protein ◽  
Smad Signaling Pathway ◽  
A Cell

ABSTRACT We identified cDNAs encoding the Xenopus Smad proteins most closely related to mammalian Smad8, and we present a functional analysis of this activity (also referred to recently as xSmad11). Misexpression experiments indicate that xSmad8(11) regulates pathways distinct from those regulated by the closely related xSmad1. Embryos that develop from eggs depleted of xSmad8(11) mRNA fail to gastrulate; instead, at the time of gastrulation, they initiate a widespread program of apoptosis, via a CPP32/caspase 3 pathway. Embryos that avoid this fate display gastrulation defects. Activation of apoptosis is rescued by expression of xSmad8(11) but not xSmad1. Our results demonstrate an embryonic requirement for Smad8(11) activity and show that a maternally derived Smad signaling pathway is required for gastrulation and for mediating a cell survival program during early embryogenesis. We suggest that xSmad8(11) functions as part of a maternally derived mechanism shown previously by others to monitor Xenopus early embryonic cell cycles.

scholarly journals The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

1993 ◽  
Vol 13 (6) ◽  
pp. 3340-3349 ◽  
Author(s):  
X Danthinne ◽  
J Seurinck ◽  
F Meulewaeter ◽  
M Van Montagu ◽  
M Cornelissen
Keyword(s):  
Tobacco Necrosis Virus ◽  
Translation System ◽  
Coding Region ◽  
Necrosis Virus ◽  
Untranslated Sequence ◽  
Virus Rna ◽  
Free Translation ◽  
Order Of Magnitude ◽  
A Cell

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

scholarly journals A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

1990 ◽  
Vol 10 (1) ◽  
pp. 146-153 ◽  
Author(s):  
K Fischman ◽  
J C Edman ◽  
G M Shackleford ◽  
J A Turner ◽  
W J Rutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Translation System ◽  
Appearance Time ◽  
Acid Protein ◽  
Mouse Tissues ◽  
Free Translation ◽  
Alternatively Spliced ◽  
A Cell ◽  
Testis Cdna ◽  
Carboxy Terminal

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

scholarly journals Chemical aminoacylation of tRNAs with fluorinated amino acids for in vitro protein mutagenesis

2010 ◽  
Vol 6 ◽  
Author(s):  
Shijie Ye ◽  
Allison Ann Berger ◽  
Dominique Petzold ◽  
Oliver Reimann ◽  
Benjamin Matt ◽  
...  
Keyword(s):  
Amino Acids ◽  
Translation System ◽  
Biologically Relevant ◽  
Fluorinated Amino Acids ◽  
Free Translation ◽  
Trna Species ◽  
A Cell ◽  
Protein Environment ◽  
Vitro Protein

This article describes the chemical aminoacylation of the yeast phenylalanine suppressor tRNA with a series of amino acids bearing fluorinated side chains via the hybrid dinucleotide pdCpA and ligation to the corresponding truncated tRNA species. Aminoacyl-tRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system. Such engineered proteins are expected to contribute to our understanding of discrete fluorines’ interaction with canonical amino acids in a native protein environment and to enable the design of fluorinated proteins with arbitrary desired properties.

Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2331-2342 ◽  
Author(s):  
Marian Takáč ◽  
Angela Witte ◽  
Udo Bläsi
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Functional Analysis ◽  
Transmembrane Domains ◽  
Class I ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Reading Frame ◽  
Double Stranded Dna ◽  
A Cell

Double-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin–endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the −1 reading frame at the 3′ end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage λ Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3…. Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.

scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

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