scholarly journals Sequence requirements for processing of proinsulin in transfected mouse pituitary AtT20 cells

1992 ◽  
Vol 286 (2) ◽  
pp. 619-622 ◽  
Author(s):  
N A Taylor ◽  
K Docherty
Keyword(s):  
Amino Acids ◽  
Model System ◽  
Proteolytic Processing ◽  
Wild Type ◽  
C Peptide ◽  
Transfected Cells ◽  
Basic Amino Acids ◽  
The Media ◽  
Mouse Pituitary ◽  
Sequence Requirements

To investigate the sequence requirements for proteolytic processing of prohormones at pairs of basic amino acids, normal and mutant proinsulins were expressed in the mouse pituitary corticotrophic cell line AtT20. The extent of processing was determined by h.p.l.c. analysis of insulin-like immunoreactivity secreted into the media of transfected cells. In this model system, normal proinsulin was efficiently processed to insulin. The mutant des-38-62-proinsulin, in which all but six amino acids of the C-peptide were deleted, was also processed to insulin but less efficiently than the wild-type. The mutant Lys64-Arg65 to Thr64-Arg65 was partially processed to insulin, while the mutant Arg31-Arg32 to Arg31-Gly32 was not processed at either site. These results indicate: (i) that a six-amino-acid spacer between the two pairs of basic amino acids in proinsulin is sufficient to permit processing at both sites; (ii) that the endoproteinase responsible for cleavage at the Lys64-Arg65 site will also recognize Thr64-Arg65; (iii) that the endoproteinase responsible for cleavage at the Arg31-Arg32 site will not recognize Arg31-Gly32; and (iv) that the change Arg31-Arg32 to Arg31-Gly32 affects processing at the Lys64-Arg65 site.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals Basic Residues in the Mason-Pfizer Monkey Virus Gag Matrix Domain Regulate Intracellular Trafficking and Capsid-Membrane Interactions

2007 ◽  
Vol 81 (17) ◽  
pp. 8977-8988 ◽  
Author(s):  
Elizabeth Stansell ◽  
Robert Apkarian ◽  
Sarka Haubova ◽  
William E. Diehl ◽  
Ewan M. Tytler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Intracellular Transport ◽  
Wild Type ◽  
Membrane Interactions ◽  
Cortical Actin ◽  
Basic Amino Acids ◽  
Gag Polyprotein ◽  
The Matrix ◽  
Arginine Residues

ABSTRACT Mason-Pfizer monkey virus (M-PMV) capsids that have assembled in the cytoplasm must be transported to and associate with the plasma membrane prior to being enveloped by a lipid bilayer during viral release. Structural studies have identified a positive-charge density on the membrane-proximal surface of the matrix (MA) protein component of the Gag polyprotein. To investigate if basic amino acids in MA play a role in intracellular transport and capsid-membrane interactions, mutants were constructed in which lysine and arginine residues (R10, K16, K20, R22, K25, K27, K33, and K39) potentially exposed on the capsid surface were replaced singly and in pairs by alanine. A majority of the charge substitution mutants were released less efficiently than the wild type. Electron microscopy of mutant Gag-expressing cells revealed four distinct phenotypes: K16A and K20A immature capsids accumulated on and budded into intracellular vesicles; R10A, K27A, and R22A capsid transport was arrested at the cellular cortical actin network, while K25A immature capsids were dispersed throughout the cytoplasm and appeared to be defective at an earlier stage of intracellular transport; and the remaining mutant (K33A and K39A) capsids accumulated at the inner surface of the plasma membrane. All mutants that released virions exhibited near-wild-type infectivity in a single-round assay. Thus, basic amino acids in the M-PMV MA define both cellular location and efficiency of virus release.

scholarly journals Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus

2006 ◽  
Vol 80 (11) ◽  
pp. 5611-5626 ◽  
Author(s):  
Jun-Young Seo ◽  
William J. Britt
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Tegument Protein ◽  
Reading Frame ◽  
Transfected Cells ◽  
Recombinant Viruses ◽  
Assembly Compartment ◽  
Sequence Requirements

ABSTRACT The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.

scholarly journals NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.

1989 ◽  
Vol 108 (4) ◽  
pp. 1237-1243 ◽  
Author(s):  
E Szczesna-Skorupa ◽  
B Kemper
Keyword(s):  
Amino Acids ◽  
Cytochrome P450 ◽  
Proteolytic Processing ◽  
Alkaline Solutions ◽  
Hydrophobic Core ◽  
Proteolytic Digestion ◽  
Basic Amino Acids ◽  
Microsomal Membranes ◽  
Transfer Signal

Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3-arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3-arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.

scholarly journals Mapping of a Region of the PA-X Protein of Influenza A Virus That Is Important for Its Shutoff Activity

2015 ◽  
Vol 89 (16) ◽  
pp. 8661-8665 ◽  
Author(s):  
Kohei Oishi ◽  
Seiya Yamayoshi ◽  
Yoshihiro Kawaoka
Keyword(s):  
Amino Acids ◽  
Influenza A Virus ◽  
Influenza A ◽  
Specific Region ◽  
Viral Mrna ◽  
Wild Type ◽  
Mrna Accumulation ◽  
Specific Domain ◽  
Basic Amino Acids ◽  
Endonuclease Domain

Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.

scholarly journals Involvement of Arg-328, Arg-334 and Arg-342 of DnaA protein in the functional interaction with acidic phospholipids

1999 ◽  
Vol 340 (2) ◽  
pp. 433-438 ◽  
Author(s):  
Yoshihiro YAMAGUCHI ◽  
Masakazu HASE ◽  
Masaki MAKISE ◽  
Shinji MIMA ◽  
Takeshi YOSHIMI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Atp Binding ◽  
Amphipathic Helix ◽  
Wild Type ◽  
Functional Interaction ◽  
Basic Amino Acids ◽  
Dnaa Protein ◽  
Acidic Amino Acids ◽  
Wild Type Protein ◽  
Acidic Phospholipids

We reported previously that three basic amino acids (Arg-360, Arg-364 and Lys-372) of DnaA protein are essential for its functional interaction with cardiolipin. In this study, we examined the effect of mutation of some basic amino acids in a potential amphipathic helix (from Lys-327 to Ile-345) of DnaA protein on this interaction. ATP binding to the mutant DnaA protein, in which Arg-328, Arg-334 and Arg-342 were changed to acidic amino acids, was less inhibited by cardiolipin than that of the wild-type protein, as was the case for mutant DnaA protein with mutations of Arg-360, Arg-364 and Lys-372. A mutant DnaA protein with mutations of all six basic amino acids showed the most resistance to the inhibition of ATP binding by cardiolipin. These results suggest that Arg-328, Arg-334 and Arg-342, like Arg-360, Arg-364 and Lys-372, are also involved in the functional interaction between DnaA protein and acidic phospholipids.

Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids

1991 ◽  
Vol 179 (3) ◽  
pp. 1181-1186 ◽  
Author(s):  
Kimimitsu Oda ◽  
Masaharu Ikeda ◽  
Emiko Tsuji ◽  
Miwa Sohda ◽  
Noboru Takami ◽  
...  
Keyword(s):  
Amino Acids ◽  
Proteolytic Cleavage ◽  
Basic Amino Acids ◽  
Sequence Requirements

Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

Does the polyamine carrier system absorb basic amino acids?

2001 ◽  
Vol 120 (5) ◽  
pp. A142-A142
Author(s):  
J GASKEY ◽  
E SEIDEL
Keyword(s):  
Amino Acids ◽  
Carrier System ◽  
Basic Amino Acids
Sign in / Sign up

Export Citation Format

Share Document