The folding capacity of the mature domain of the dual-targeted plant tRNA nucleotidyltransferase influences organelle selection

2013 ◽  
Vol 453 (3) ◽  
pp. 401-412 ◽  
Author(s):  
Matthew Leibovitch ◽  
Daniela Bublak ◽  
Pamela J. Hanic-Joyce ◽  
Bodo Tillmann ◽  
Nadine Flinner ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Physicochemical Properties ◽  
Single Gene ◽  
Transit Peptide ◽  
Intracellular Distribution ◽  
Peptide Sequence ◽  
Mature Protein ◽  
Nuclear Targeting ◽  
Mature Domain ◽  
Cellular Compartments

tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3′-termini. Therefore this enzymatic activity is found in all cellular compartments, namely the nucleus, cytoplasm, plastids and mitochondria, in which tRNA synthesis or translation occurs. A single gene codes for tRNA-NT in plants, suggesting a complex targeting mechanism. Consistent with this, distinct signals have been proposed for plastidic, mitochondrial and nuclear targeting. Our previous research has shown that in addition to N-terminal targeting information, the mature domain of the protein itself modifies targeting to mitochondria and plastids. This suggests the existence of an as yet unknown determinate for the distribution of dual-targeted proteins between these two organelles. In the present study, we explore the enzymatic and physicochemical properties of tRNA-NT variants to correlate the properties of the enzyme with the intracellular distribution of the protein. We show that alteration of tRNA-NT stability influences its intracellular distribution due to variations in organelle import capacities. Hence the fate of the protein is determined not only by the transit peptide sequence, but also by the physicochemical properties of the mature protein.

scholarly journals A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare)

1996 ◽  
Vol 313 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Tine THORBJØRNSEN ◽  
Per VILLAND ◽  
Leszek A. KLECZKOWSKI ◽  
Odd-Arne OLSEN
Keyword(s):  
Hordeum Vulgare ◽  
Genomic Library ◽  
Single Gene ◽  
Starch Synthesis ◽  
Transit Peptide ◽  
Small Subunit ◽  
Peptide Sequence ◽  
Subunit Gene ◽  
Starchy Endosperm ◽  
Adp Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepsF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5ʹ ends. In blps14, the unique 5ʹ end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5ʹ end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5ʹ ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5ʹ-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing.

scholarly journals Overexpression of an EaZIP gene devoid of transit peptide sequence induced leaf variegation in tobacco

PLoS ONE ◽  
2017 ◽  
Vol 12 (4) ◽  
pp. e0175995 ◽  
Author(s):  
Xiayu Guan ◽  
Zhijian Li ◽  
Zhiliang Zhang ◽  
Xiangying Wei ◽  
Jiahua Xie ◽  
...  
Keyword(s):  
Transit Peptide ◽  
Peptide Sequence ◽  
Leaf Variegation

scholarly journals Regulation of Poly(ADP-ribose) Polymerase-1-dependent Gene Expression through Promoter-directed Recruitment of a Nuclear NAD+ Synthase

2012 ◽  
Vol 287 (15) ◽  
pp. 12405-12416 ◽  
Author(s):  
Tong Zhang ◽  
Jhoanna G. Berrocal ◽  
Jie Yao ◽  
Michelle E. DuMond ◽  
Raga Krishnakumar ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Target Gene ◽  
Target Genes ◽  
Enzymatic Activities ◽  
Gene Promoters ◽  
Protein Coding ◽  
Photon Excitation ◽  
Gene Sets ◽  
Cellular Compartments ◽  
Parp 1

NMNAT-1 and PARP-1, two key enzymes in the NAD+ metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters where it produces NAD+ to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independently of NAD+ production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD+ in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent poly(ADP-ribosyl)ation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters.

Differential roles of two DDX17 isoforms in the formation of membraneless organelles

2020 ◽  
Vol 168 (1) ◽  
pp. 33-40
Author(s):  
Yuya Hirai ◽  
Eisuke Domae ◽  
Yoshihiro Yoshikawa ◽  
Keizo Tomonaga
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Rna Helicase ◽  
Intracellular Distribution ◽  
Amino Acid Sequences ◽  
Nucleolar Localization ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Dead Box ◽  
Additional Amino Acid

Abstract The RNA helicase, DDX17 is a member of the DEAD-box protein family. DDX17 has two isoforms: p72 and p82. The p82 isoform has additional amino acid sequences called intrinsically disordered regions (IDRs), which are related to the formation of membraneless organelles (MLOs). Here, we reveal that p72 is mostly localized to the nucleoplasm, while p82 is localized to the nucleoplasm and nucleoli. Additionally, p82 exhibited slower intranuclear mobility than p72. Furthermore, the enzymatic mutants of both p72 and p82 accumulate into the stress granules. The enzymatic mutant of p82 abolishes nucleolar localization of p82. Our findings suggest the importance of IDRs and enzymatic activity of DEAD-box proteins in the intracellular distribution and formation of MLOs.

The role of an urban park's tree stand in shaping the enzymatic activity, glomalin content and physicochemical properties of soil

2020 ◽  
Vol 741 ◽  
pp. 140446 ◽  
Author(s):  
Joanna Lemanowicz ◽  
Samir A. Haddad ◽  
Agata Bartkowiak ◽  
Robert Lamparski ◽  
Piotr Wojewódzki
Keyword(s):  
Enzymatic Activity ◽  
Physicochemical Properties ◽  
Tree Stand ◽  
Properties Of Soil

Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding

Parasitology ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 149-157 ◽  
Author(s):  
B.-K. NA ◽  
J.-M. KANG ◽  
H.-I. CHEUN ◽  
S.-H. CHO ◽  
S.-U. MOON ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Transmembrane Domain ◽  
Cathepsin L ◽  
Active Form ◽  
Protozoan Parasite ◽  
Biochemical Properties ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Mature Domain

SUMMARYCryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.

scholarly journals Intracellular Distribution of 5' Nucleotidase in Rat Liver

1958 ◽  
Vol 4 (4) ◽  
pp. 373-376 ◽  
Author(s):  
Gaston de Lamirande ◽  
Claude Allard ◽  
Antonio Cantero
Keyword(s):  
Enzymatic Activity ◽  
Rat Liver ◽  
Biochemical Analysis ◽  
Inorganic Phosphorus ◽  
Intracellular Distribution ◽  
Differential Centrifugation ◽  
Nucleotidase Activity ◽  
Rat Liver Cells ◽  
Cell Fractions

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg++ ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.

scholarly journals Stromal Processing Peptidase Binds Transit Peptides and Initiates Their Atp-Dependent Turnover in Chloroplasts

1999 ◽  
Vol 147 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Stefan Richter ◽  
Gayle K. Lamppa
Keyword(s):  
Binding Assay ◽  
Specific Binding ◽  
Transit Peptide ◽  
Binding Motif ◽  
Mature Protein ◽  
Sequence Of Events ◽  
Mitochondrial Processing Peptidase ◽  
Transit Peptides ◽  
Processing Peptidase ◽  
Zinc Binding Motif

A stromal processing peptidase (SPP) cleaves a broad range of precursors targeted to the chloroplast, yielding proteins for numerous biosynthetic pathways in different compartments. SPP contains a signature zinc-binding motif, His-X-X-Glu-His, that places it in a metallopeptidase family which includes the mitochondrial processing peptidase. Here, we have investigated the mechanism of cleavage by SPP, a late, yet key event in the import pathway. Recombinant SPP removed the transit peptide from a variety of precursors in a single endoproteolytic step. Whereas the mature protein was immediately released, the transit peptide remained bound to SPP. SPP converted the transit peptide to a subfragment form that it no longer recognized. We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release. A stable interaction between SPP and an intact transit peptide was directly demonstrated using a newly developed binding assay. Unlike recombinant SPP, a chloroplast extract rapidly degraded both the transit peptide and subfragment. A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent. Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

scholarly journals Human plasma fibronectin. Demonstration of structural differences between the A- and B-chains in the III CS region

1991 ◽  
Vol 274 (3) ◽  
pp. 731-738 ◽  
Author(s):  
T Tressel ◽  
J B McCarthy ◽  
J Calaycay ◽  
T D Lee ◽  
K Legesse ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Single Gene ◽  
Cell Types ◽  
Binding Domain ◽  
Peptide Sequence ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Binding Domains ◽  
A Chain

Fibronectins are a class of cell-adhesion proteins produced from a single gene. The soluble plasma form is synthesized by hepatocytes and the insoluble cellular form by fibroblasts and other cell types. The proteins possess multiple binding domains for macromolecules including collagen, fibrin and heparin along with at least one cell-binding domain. Cellular as well as plasma fibronectins are dimers of similar but not identical polypeptides. Their differences are the result of internal amino acid sequence variability due to alternative RNA splicing in at least three regions (ED-A, ED-B and III CS). We have been studying this polymorphism at the protein level in plasma fibronectin (pFn). Cathepsin D-digested pFn applied to a heparin-agarose column and eluted with an NaCl stepwise gradient (0.1 M, 0.25 M and 0.5 M) released two polypeptides (75 kDa and 65 kDa) in the 0.5 M-NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the C-terminal heparin-binding domain) and AHB-3 (specific for the III CS domain) suggest that both peptides contain the C-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the III CS region. These two polypeptides (75 kDa and 65 kDa) were digested with trypsin, and the resulting peptides were analyzed by fast-atom-bombardment mass spectrometry and compared with the known cDNA-derived peptide sequence. Peptides that were unique to the III CS region were further characterized by micro sequence analysis. The 75 kDa fragment is derived from the A-chain and contains the III CS region (89 amino acid residues) along with the C-terminal heparin-binding (Hep-2) domain and the fibrin-binding (Fib-2) domain. A single galactosamine-based carbohydrate group was detected at Thr-73/74 of the III CS region present in the 75 kDa fragment. The 65 kDa fragment is derived from the B-chain and lacks the entire III CS region but does contain the Hep-2 and Fib-2 domains.

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