scholarly journals Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

1992 ◽  
Vol 12 (12) ◽  
pp. 5652-5658 ◽  
Author(s):  
A M Rose ◽  
P B Joyce ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Amino Acids ◽  
Nuclear Periphery ◽  
Immunofluorescent Staining ◽  
Coding Region ◽  
Nuclear Targeting ◽  
Subnuclear Localization ◽  
Amino Terminal ◽  
Linear Sequence ◽  
Trna Methyltransferase ◽  
Terminal Amino

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei

1992 ◽  
Vol 12 (12) ◽  
pp. 5652-5658
Author(s):  
A M Rose ◽  
P B Joyce ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Amino Acids ◽  
Nuclear Periphery ◽  
Immunofluorescent Staining ◽  
Coding Region ◽  
Nuclear Targeting ◽  
Subnuclear Localization ◽  
Amino Terminal ◽  
Linear Sequence ◽  
Trna Methyltransferase ◽  
Terminal Amino

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

scholarly journals THE AMINO-TERMINAL AMINO ACIDS OF PAROTIN

1960 ◽  
Vol 7 (3) ◽  
pp. 175-180
Author(s):  
YUKIHO KUBOTA
Keyword(s):  
Amino Acids ◽  
Amino Terminal ◽  
Terminal Amino

Coronin localizes to leading edges and is involved in cell spreading and lamellipodium extension in vertebrate cells

1999 ◽  
Vol 112 (17) ◽  
pp. 2833-2842 ◽  
Author(s):  
M. Mishima ◽  
E. Nishida
Keyword(s):  
Amino Acids ◽  
Cell Spreading ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Cellular Slime Mold ◽  
Cell Periphery ◽  
Terminal Amino ◽  
Wd Repeat ◽  
Leading Edges

Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. We cloned a Xenopus homolog of coronin (Xcoronin) and examined its actin-binding properties, subcellular localization and possible functions. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sedimented with F-actin in vitro. The WD repeat domain (residues 64–299) alone did not have any affinity for F-actin. Anti-Xcoronin antibodies reacted specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells revealed that Xcoronin is present in the cytoplasm and concentrated in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin became concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery was resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolished the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts was concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64–299), but not of full-length Xcoronin, significantly decreased the rate of cell spreading after replating and markedly inhibited lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading.

Functional Analysis of a Peptide Sequence (Thr323 - Cys337) In ADAMTS13 Disintegrin-Like Domain Revealed Two Discontinuous Sequences Involved In the Interaction with VWF

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2200-2200
Author(s):  
Atsuko Igari ◽  
Takanori Moriki ◽  
Terumichi Nakagawa ◽  
Yusuke Yamaguchi ◽  
Mitsuru Murata
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Synthetic Peptides ◽  
Inhibitory Effect ◽  
Peptide Sequence ◽  
Recent Analysis ◽  
Inhibitory Effects ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Carboxyl Terminal

Abstract Abstract 2200 ADAMTS13 specifically cleaves multimeric von Willebrand factor (VWF) into smaller molecules to reduce its high reactivity with platelets. The disintegrin-like (D) domain, adjacent to the catalytic domain of ADAMTS13, plays an important role in the process of VWF cleavage. In this study, we aimed to elucidate critical peptide sequences in D-domain involved in the interaction with VWF. A series of partially overlapping peptide sequences, approximately 20 amino acids in length, covering the D-domain, were synthesized and the inhibitory effects on the catalytic activity of plasma ADAMTS13 was examined using FRETS-VWF73 assay. Consequently, some synthetic peptides were selected and the minimal length necessary for the inhibitory effect was determined as TFAREHLDMCQALSC (peptide323-337). Removal of the amino-terminal threonine diminished the inhibitory effect moderately, although deletion of the carboxyl-terminal cysteine abolished it completely. According to the amino acids alignment of ADAMTS family, this peptide sequence is not conserved, highlighting the specific role in the interaction with its substrate. From the recent analysis of crystal structure, amino-terminal half of the peptide323-337, TFAREHL (323-329), was disordered and designated as the variable (V) loop, which creates one of VWF-binding exosites (Akiyama, et al. Proc Natl Acad Sci USA. 2009; 106:19274-9). We hypothesized that the amino-terminal amino acids of the peptide323-337 contribute to VWF binding, whereas the carboxyl-terminal amino acids allow the structural stability of the peptide conformation. To evaluate the effect of carboxyl-terminal cysteine at 337, other synthetic peptides with alanine, serine, glycine or phenylalanine instead of the cysteine (C337A, C337S, C337G, or C337F) were tested about their inhibitory effects on the catalytic activity. Interestingly, C337A, C337S, C337G peptides exhibited slightly weaker inhibitory effects on VWF73 catalysis, although C337F peptide showed stronger inhibition than wild-type sequence, suggesting that the residue 337 regulates the characteristics of the peptide323-337. From the results of peptide screening, the amino- and carboxyl-terminal amino acids of the peptide323-337, TFAREHLDMCQALSC, likely play key roles in the inhibitory effects; therefore, the middle part of the sequence, HLDMC, was replaced by 5 alanines (AAAAA) or reversed sequence CMDLH. Surprisingly, the converted peptides still retained the equivalent level of inhibitory effects, indicating both sides of the amino- and carboxyl-terminal amino acids were especially significant in the interaction with VWF. In conclusion, we characterized the peptide sequence, TFAREHLDMCQALSC (323-337), in D-domain. The peptide clearly inhibited the cleavage of VWF73 and the both sides of amino- and carboxyl-terminal amino acids seemed especially important. The peptide sequence is supposed to bind to VWF for the precise cleavage in the process of proteolysis. By modifying this peptide sequence, such variant ADAMTS13 as gain-of-function recombinants might be developed, leading to an alternative anti-thrombotic drug. Disclosures: No relevant conflicts of interest to declare.

The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras

1988 ◽  
Vol 8 (9) ◽  
pp. 3960-3963
Author(s):  
J E Buss ◽  
C J Der ◽  
P A Solski
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Myristic Acid ◽  
Amino Terminus ◽  
Directed Mutagenesis ◽  
Ras Protein ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Crucial Part

We have used oligonucleotide-directed mutagenesis to replace the N-terminal amino acids of p21v-ras with residues which mimic the amino terminus of p60v-src. p21v-ras protein possessing only the first five amino acids of p60src was not myristylated, while substitution of residue 6 (serine) produced a protein p21(GSSKS) which incorporated [3H]myristic acid that was stable to hydroxylamine, sensitive to inhibitors of protein synthesis, and found in both the normally nonacylated precursor and mature forms of p21(GSSKS). This defines the minimum framework of the p60v-src myristylation signal (glycine 2 and serine 6) and identifies serine 6 as a crucial part of that signal for myristylation of a protein in vivo.

DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes

1987 ◽  
Vol 7 (5) ◽  
pp. 1978-1983
Author(s):  
A Tanaka ◽  
C P Gibbs ◽  
R R Arthur ◽  
S K Anderson ◽  
H J Kung ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Sequence Divergence ◽  
Terminal Region ◽  
Specific Substrate ◽  
Coding Region ◽  
Exon 2 ◽  
Amino Terminal ◽  
Src Gene ◽  
Sequence Encoding

We sequenced the 5'-coding region of the human c-src gene, exons 2 through 5, corresponding to one-third of the human c-src protein consisting of 536 amino acids. Sequence analysis of the src type of protein kinases revealed that the amino-terminal region encoded by exon 2 contains sequences specific for the src proteins and raised the possibility that this region is involved in the recognition of a src-specific substrate(s) or receptor(s).

scholarly journals N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae.

1989 ◽  
Vol 109 (4) ◽  
pp. 1411-1419 ◽  
Author(s):  
J M Li ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Indirect Immunofluorescence ◽  
Trna Modification ◽  
Mitochondrial Targeting ◽  
Nuclear Targeting ◽  
Cytoplasmic Staining ◽  
Immunofluorescence Studies ◽  
Trna Methyltransferase ◽  
Terminal Amino ◽  
Beta Galactosidase

The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs. The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs. Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus. Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane. There is no obvious cytoplasmic staining in these overproducing strains. Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme. Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.

scholarly journals DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes.

1987 ◽  
Vol 7 (5) ◽  
pp. 1978-1983 ◽  
Author(s):  
A Tanaka ◽  
C P Gibbs ◽  
R R Arthur ◽  
S K Anderson ◽  
H J Kung ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Sequence Divergence ◽  
Terminal Region ◽  
Specific Substrate ◽  
Coding Region ◽  
Exon 2 ◽  
Amino Terminal ◽  
Src Gene ◽  
Sequence Encoding

We sequenced the 5'-coding region of the human c-src gene, exons 2 through 5, corresponding to one-third of the human c-src protein consisting of 536 amino acids. Sequence analysis of the src type of protein kinases revealed that the amino-terminal region encoded by exon 2 contains sequences specific for the src proteins and raised the possibility that this region is involved in the recognition of a src-specific substrate(s) or receptor(s).

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