The Amino-Terminal Domain of Chloroplast Hsp93 Is Important for Its Membrane Association and Functions in Vivo

The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation.

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A mitogen- and anisomycin-stimulated kinase phosphorylates HMG-14 in its basic amino-terminal domain in vivo and on isolated mononucleosomes.

The EMBO Journal ◽

10.1002/j.1460-2075.1994.tb06774.x ◽

1994 ◽

Vol 13 (19) ◽

pp. 4524-4535 ◽

Cited By ~ 30

Author(s):

M.J. Barratt ◽

C.A. Hazzalin ◽

N. Zhelev ◽

L.C. Mahadevan

Keyword(s):

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

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Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

Journal of Bacteriology ◽

10.1128/jb.01705-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2279-2285 ◽

Cited By ~ 13

Author(s):

Georgeta N. Basturea ◽

Maria D. Bodero ◽

Mario E. Moreno ◽

George P. Munson

Keyword(s):

Dna Binding ◽

Amino Terminus ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal ◽

Amino Terminal Domain ◽

Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

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BBK07, a Dominant In Vivo Antigen of Borrelia burgdorferi, Is a Potential Marker for Serodiagnosis of Lyme Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00301-09 ◽

2009 ◽

Vol 16 (11) ◽

pp. 1569-1575 ◽

Cited By ~ 8

Author(s):

Adam S. Coleman ◽

Utpal Pal

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Amino Acid Identity ◽

Amino Terminal ◽

Terminal Domain ◽

Potential Marker ◽

Mammalian Infection ◽

Amino Terminal Domain

ABSTRACT One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.

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Protein product of proto-oncogene c-mil

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.739-744.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 739-744

Author(s):

T Patschinsky ◽

B Schroeer ◽

K Bister

Keyword(s):

Molecular Weight ◽

Cellular Protein ◽

Protein Product ◽

Homologous Proteins ◽

Molecular Weights ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain ◽

Relationship Of

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.

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Identification of New In Vivo TonB-FepA Rendezvous Sites

10.1101/2021.12.08.471779 ◽

2021 ◽

Author(s):

Kathleen Postle ◽

Kelvin Kho ◽

Michael Gresock ◽

Joydeep Ghosh ◽

Ray Larsen

Keyword(s):

Outer Membrane ◽

Cytoplasmic Membrane ◽

Protonmotive Force ◽

Amphipathic Helix ◽

Amino Terminal ◽

Terminal Domain ◽

Tonb System ◽

Carboxy Terminal ◽

Amino Terminal Domain

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

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Protein product of proto-oncogene c-mil.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.739 ◽

1986 ◽

Vol 6 (2) ◽

pp. 739-744 ◽

Cited By ~ 6

Author(s):

T Patschinsky ◽

B Schroeer ◽

K Bister

Keyword(s):

Molecular Weight ◽

Cellular Protein ◽

Protein Product ◽

Homologous Proteins ◽

Molecular Weights ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain ◽

Relationship Of

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.

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ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1203 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1203-1209 ◽

Cited By ~ 104

Author(s):

J Horiuchi ◽

N Silverman ◽

G A Marcus ◽

L Guarente

Keyword(s):

Activation Domains ◽

Amino Terminal ◽

Terminal Domain ◽

Trimeric Complex ◽

Nonoverlapping Domains ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

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Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5380 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5380-5391 ◽

Cited By ~ 39

Author(s):

Enrique Castaño ◽

Yelena Kleyner ◽

Brian David Dynlacht

Keyword(s):

Mammalian Cells ◽

Kinase Inhibitor ◽

Point Mutations ◽

Growth Suppression ◽

Kinase Substrate ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

ABSTRACT The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki ) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.

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