Biomedical subjects

Saibal Chatterjee

Publications and source records attributed to Saibal Chatterjee.

6 recordsLinked to original sources

PubMed2006

An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria.

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.

Allosteric Site↗

PubMed2006

A bifunctional tRNA import receptor from Leishmania mitochondria.

In kinetoplastid protozoa, import of cytosolic tRNAs into mitochondria occurs through tRNAs interacting with membrane-bound proteins, the identities of which are unknown. The inner membrane RNA import complex of Leishmania tropica contains multiple proteins and is active for import in vitro. RIC1, the largest subunit of this complex, is structurally homologous to the conserved alpha subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli. Antisense-mediated knockdown of RIC1/F1alpha in Leishmania resulted in depletion of several mitochondrial tRNAs belonging to distinct subsets (types I and II) that interact cooperatively or antagonistically within the import complex. The knockdown-induced defect in import of type I tRNAs was rectified in a reconstituted system by purified RIC1/F1alpha alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1alpha formed stable complexes with type I, but not type II, tRNAs through the cooperation of its nucleotide binding and C-terminal domains. Thus, RIC1/F1alpha is a type I tRNA import receptor. As expected of a bifunctional protein, RIC1/F1alpha is shared by both the import complex and by respiratory complex V. Alternative use of ancient respiratory proteins may have been an important step in the evolution of tRNA import.

Animals↗

PubMed2003

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria.

Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA(Tyr) (Type I) transiently stimulates the rate of entry of tRNA(Ile) (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA(Ile). Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA(Tyr) to the D domain, and those of tRNA(Ile) to the variable region-T domain (V-T region), indicating that both activities originate from a single RNA-receptor interaction. A third tRNA, human tRNA(Lys), is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.

Allosteric Regulation↗

PubMed2003

Mitochondrial differentiation in kinetoplastid protozoa: a plethora of RNA controls.

Differentiation of kinetoplastid protozoa during their complex life cycles is accompanied by stepwise changes in mitochondrial functions. Recent studies have begun to reveal multilevel post-transcriptional regulatory mechanisms by which the expression of the nuclear and mitochondrially encoded components of respiratory enzymes is coordinated, as well as the identities of some general and gene-specific factors controlling mitochondrial differentiation.

Animals↗

PubMed2003

"Ping-pong" interactions between mitochondrial tRNA import receptors within a multiprotein complex.

The mitochondrial genomes of a wide variety of species contain an insufficient number of functional tRNA genes, and translation of mitochondrial mRNAs is sustained by import of nucleus-encoded tRNAs. In Leishmania, transfer of tRNAs across the inner membrane can be regulated by positive and negative interactions between them. To define the factors involved in such interactions, a large multisubunit complex (molecular mass, approximately 640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania, consisting of approximately 130-A particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP- and proton motive force-dependent transfer of Leishmania tRNA(Tyr) as well as of oligoribonucleotides containing the import signal YGGYAGAGC. Moreover, allosteric interactions between tRNA(Tyr) and tRNA(Ile) were observed in the RNA import complex-reconstituted system, indicating the presence of primary and secondary tRNA binding sites within the complex. By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNA(Ile) to a 21-kDa component of the complex is dependent upon tRNA(Tyr), while binding of tRNA(Tyr) to a 45-kDa component is inhibited by tRNA(Ile). This "ping-pong" mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation.

Allosteric Site↗

PubMed2002

Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane.

A large number of cytoplasmic tRNAs are imported into the kinetoplast-mitochondrion of Leishmania by a receptor-mediated process. To identify the sequences recognized by import receptors, mitochondria were incubated with a combinatorial RNA library. Repeated cycles of amplification of the imported sequences (SELEX) resulted in rapid selection of several import aptamers containing sequence motifs present in the anticodon arm, the D arm, the V-T region, and acceptor stem of known tRNAs, confirming or suggesting the presence of import signals in these domains. As predicted, truncated derivatives of tRNA(Ile)(UAU) containing the D arm or the V-T region were imported in vitro. Four aptamers were studied in detail. All were imported in vitro as well as in transiently transfected cells, using the same pathway as tRNA, but their individual import efficiencies were different. Two types of aptamers were discernible: the A arm and D arm homologues (type I), which were efficiently transferred across the inner mitochondrial membrane, and the V-T homologues (type II), which were not. Remarkably, subnanomolar concentrations of type I RNAs stimulated the entry of type II RNAs into the matrix, whereas type II RNAs inhibited inner membrane transfer of type I RNAs. Moreover, tRNA(Tyr)(GUA) and tRNA(Ile)(UAU) interacted with one another as type I and type II, respectively. Such cooperative and antagonistic interactions may allow the use of a limited number of receptors to recognize a large number of tRNAs of variable affinity and enable the maintenance of a properly balanced tRNA pool for mitochondrial translation.

Animals↗