scholarly journals The Spliced Leader RNA Silencing (SLS) Pathway in Trypanosoma brucei Is Induced by Perturbations of Endoplasmic Reticulum, Golgi Complex, or Mitochondrial Protein Factors: Functional Analysis of SLS-Inducing Kinase PK3

mBio ◽  
2021 ◽  
Author(s):  
Uthman Okalang ◽  
Bar Mualem Bar-Ner ◽  
K. Shanmugha Rajan ◽  
Nehemya Friedman ◽  
Saurav Aryal ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Trypanosoma Brucei ◽  
Golgi Complex ◽  
Rna Silencing ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Sulfhydryl Oxidase ◽  
Mitochondrial Protein Import ◽  
Spliced Leader ◽  
Quiescin Sulfhydryl Oxidase

In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS.

scholarly journals The spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei is induced by perturbations of endoplasmic reticulum, Golgi, or mitochondrial proteins factors and functional analysis of SLS inducing kinase, PK3

2021 ◽  
Author(s):  
Uthman Okalang ◽  
Bar Mualem Bar-Ner ◽  
K. Shanmugha Rajan ◽  
Nehemya Friedman ◽  
Saurav Aryal ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Trypanosoma Brucei ◽  
Rna Silencing ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Serine Threonine Kinase ◽  
Spliced Leader ◽  
Quiescin Sulfhydryl Oxidase ◽  
Spliced Leader Rna

ABSTRACTIn the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5’ exon derived from the spliced leader RNA (SL RNA). Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA binding protein TRF4, leading to the shut-off of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi-localized quiescin sulfhydryl oxidase (QSOX1). Most strikingly, silencing of Rhomboid-like 1(TIMRHOM1) involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structure of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.

Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

2000 ◽  
Vol 276 (3) ◽  
pp. 1028-1034 ◽  
Author(s):  
Khaleque Md. Abdul ◽  
Kazutoyo Terada ◽  
Masato Yano ◽  
Michael T. Ryan ◽  
Illo Streimann ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Protein Import ◽  
Cultured Cells ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
Tom Complex

Functional analysis of mitochondrial protein import in yeast

1988 ◽  
Vol 36 (3) ◽  
pp. 275-287 ◽  
Author(s):  
Scott M. Glaser ◽  
Cynthia E. Trueblood ◽  
Lori K. Dircks ◽  
Robert O. Poyton ◽  
Michael G. Cumsky
Keyword(s):  
Functional Analysis ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import

scholarly journals A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import

2016 ◽  
Vol 214 (4) ◽  
pp. 417-431 ◽  
Author(s):  
Ajay Ramesh ◽  
Valentina Peleh ◽  
Sonia Martinez-Caballero ◽  
Florian Wollweber ◽  
Frederik Sommer ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Voltage Gating ◽  
Intermembrane Space ◽  
Sulfhydryl Oxidase ◽  
Mitochondrial Protein Import ◽  
Conducting Channel ◽  
Oxidoreductase Activity

Tim17 is a central, membrane-embedded subunit of the mitochondrial protein import machinery. In this study, we show that Tim17 contains a pair of highly conserved cysteine residues that form a structural disulfide bond exposed to the intermembrane space (IMS). This disulfide bond is critical for efficient protein translocation through the TIM23 complex and for dynamic gating of its preprotein-conducting channel. The disulfide bond in Tim17 is formed during insertion of the protein into the inner membrane. Whereas the import of Tim17 depends on the binding to the IMS protein Mia40, the oxidoreductase activity of Mia40 is surprisingly dispensable for Tim17 oxidation. Our observations suggest that Tim17 can be directly oxidized by the sulfhydryl oxidase Erv1. Thus, import and oxidation of Tim17 are mediated by the mitochondrial disulfide relay, though the mechanism by which the disulfide bond in Tim17 is formed differs considerably from that of soluble IMS proteins.

scholarly journals Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase

2008 ◽  
Vol 183 (2) ◽  
pp. 195-202 ◽  
Author(s):  
Diana Stojanovski ◽  
Dusanka Milenkovic ◽  
Judith M. Müller ◽  
Kipros Gabriel ◽  
Agnes Schulze-Specking ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Ternary Complex ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Intermediate Step ◽  
Intermembrane Space ◽  
Sulfhydryl Oxidase ◽  
Mitochondrial Protein Import ◽  
Substrate Protein ◽  
Mitochondrial Intermembrane Space

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.

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