scholarly journals The C-terminal Region of Mitochondrial Single-subunit RNA Polymerases Contains Species-specific Determinants for Maintenance of Intact Mitochondrial Genomes

2002 ◽  
Vol 13 (7) ◽  
pp. 2245-2255
Author(s):  
Thomas Lisowsky ◽  
Detlef Wilkens ◽  
Torsten Stein ◽  
Boris Hedtke ◽  
Thomas Börner ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Rna Polymerases ◽  
Mitochondrial Genomes ◽  
Mitochondrial Rna ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Stable Maintenance ◽  
Species Specific

Functional conservation of mitochondrial RNA polymerases was investigated in vivo by heterologous complementation studies in yeast. It turned out that neither the full-length mitochondrial RNA polymerase of Arabidopsis thaliana, nor a set of chimeric fusion constructs from plant and yeast RNA polymerases can substitute for the yeast mitochondrial core enzyme Rpo41p when expressed in Δrpo41 yeast mutants. Mitochondria from mutant cells, expressing the heterologous mitochondrial RNA polymerases, were devoid of any mitochondrial genomes. One important exception was observed when the carboxyl-terminal domain of Rpo41p was exchanged with its plant counterpart. Although this fusion protein could not restore respiratory function, stable maintenance of mitochondrial petite genomes (ρ−)− was supported. A carboxyl-terminally truncated Rpo41p exhibited a comparable activity, in spite of the fact that it was found to be transcriptionally inactive. Finally, we tested the carboxyl-terminal domain for complementation intrans. For this purpose the last 377 amino acid residues of yeast mitochondrial Rpo41p were fused to its mitochondrial import sequence. Coexpression of this fusion protein with C-terminally truncated Rpo41p complemented the Δrpo41 defect. These data reveal the importance of the carboxyl-terminal extension of Rpo41p for stable maintenance of intact mitochondrial genomes and for distinct species-specific intramolecular protein–protein interactions.

scholarly journals Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

1996 ◽  
Vol 7 (2) ◽  
pp. 261-272 ◽  
Author(s):  
U Gottwald ◽  
R Brokamp ◽  
I Karakesisoglou ◽  
M Schleicher ◽  
A A Noegel
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
Dictyostelium Discoideum ◽  
Direct Interaction ◽  
Actin Binding ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

scholarly journals In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

1997 ◽  
Vol 8 (1) ◽  
pp. 33-46 ◽  
Author(s):  
N L Schlaich ◽  
M Häner ◽  
A Lustig ◽  
U Aebi ◽  
E C Hurt
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Full Length ◽  
Yeast Cells ◽  
Heptad Repeat ◽  
Nuclear Pores ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

scholarly journals Functional Characterization of a Nup159p-containing Nuclear Pore Subcomplex

1998 ◽  
Vol 9 (12) ◽  
pp. 3475-3492 ◽  
Author(s):  
Naı̈ma Belgareh ◽  
Christine Snay-Hodge ◽  
Fabien Pasteau ◽  
Suzanne Dagher ◽  
Charles N. Cole ◽  
...  
Keyword(s):  
Protein Import ◽  
Functional Characterization ◽  
Nuclear Pore ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Cytoplasmic Face ◽  
Rna Export ◽  
Carboxyl Terminal

Nup159p/Rat7p is an essential FG repeat–containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+RNA export and NPC distribution. A detailed structural–functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Δ108mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Δ108 cells grown at 37°C, a temperature at which the Nup82Δ108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Δ108and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)+RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.

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

1995 ◽  
Vol 15 (3) ◽  
pp. 1203-1209 ◽  
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.

scholarly journals Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations.

Genetics ◽  
1995 ◽  
Vol 140 (4) ◽  
pp. 1223-1233
Author(s):  
M L West ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Phosphorylation Sites ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Rna Polymerase Ii Ctd

Abstract The carboxyl-terminal domain (CTD) of the RNA polymerase II largest subunit plays an essential but poorly understood role in transcription. The CTD is highly phosphorylated in vivo and this modification may be important in the transition from transcription initiation to elongation. We report here the development of a strategy for creating novel yeast CTDs. We have used this approach to show that the minimum viable CTD in yeast contains eight consensus (Tyr1Ser2Pro3Thr4Ser5Pro6Ser7) heptapeptide repeats. Substitution of alanine or glutamate for serines in positions two or five is lethal. In addition, changing tyrosine in position one to phenylalanine is lethal. The effects of mutations that alter potential phosphorylation sites are consistent with a requirement for CTD phosphorylation in vivo.

Sign in / Sign up

Export Citation Format

Share Document