RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy

Kim, Hag Dong; Kong, Eun Bin; Kim, Yong Joong; Chang, Jin Soo; Kim, Joon

doi:10.1038/cddis.2017.204

Cited by 24 publications

(19 citation statements)

References 58 publications

(65 reference statements)

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“…This approach has been used to investigate RACK1’s translation-specific functions (8, 10, 15, 25). Similar to earlier studies of RACK1 (8, 26), analysis of LACK’s interaction with Leishmania ribosomes (13) predicts that replacement of LACK’s noncanonical orthologous motif, RDG, with DDE will disrupt LACK’s ribosomal interaction. We therefore generated an L.…”

Section: Discussionsupporting

confidence: 64%

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs CytochromecOxidase Subunit Expression inLeishmania major

Cardenas

Sylvester

Cao

et al. 2019

mSphere

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During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK, a gene that encodes the ribosome-associated scaffold protein, LACK (Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACKR34D35G36→LACKD34D35E36) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACKD34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome. IMPORTANCE Leishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani. The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK’s putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK’s interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK’s ribosomal interactions as a potential therapeutic target.

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Section: Discussionsupporting

confidence: 64%

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs CytochromecOxidase Subunit Expression inLeishmania major

Cardenas

Sylvester

Cao

et al. 2019

mSphere

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“…While the vast majority of studies exploring core autophagy regulation via ATG proteins has focused mainly on their molecular actions, less attention has been given to mechanisms ensuring their adequate levels of production. More elaborate insight has been obtained with regard to transcriptional control of ATG transcripts , though only a small handful of studies have addressed aspects of translational regulation of ATG proteins . Importantly, the highly dynamic nature of autophagy, its importance in stress response and the potentially detrimental consequences of its dysregulation all underscore the necessity for its tightly coordinated control at multiple levels.…”

Section: Discussionmentioning

confidence: 99%

eIF 5A is required for autophagy by mediating ATG 3 translation

et al. 2018

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Autophagy is an essential catabolic process responsible for recycling of intracellular material and preserving cellular fidelity. Key to the autophagy pathway is the ubiquitin-like conjugation system mediating lipidation of Atg8 proteins and their anchoring to autophagosomal membranes. While regulation of autophagy has been characterized at the level of transcription, protein interactions and post-translational modifications, its translational regulation remains elusive. Here we describe a role for the conserved eukaryotic translation initiation factor 5A (eIF5A) in autophagy. Identified from a high-throughput screen, we find that eIF5A is required for lipidation of LC3B and its paralogs and promotes autophagosome formation. This feature is evolutionarily conserved and results from the translation of the E2-like ATG3 protein. Mechanistically, we identify an amino acid motif in ATG3 causing eIF5A dependency for its efficient translation. Our study identifies eIF5A as a key requirement for autophagosome formation and demonstrates the importance of translation in mediating efficient autophagy.

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“…In addition to ribosomes lacking RPs being present in normal tissues, they are also associated with various cancers. The protein RACK1/Asc1 affects ribosomal activity in multiple contexts, and ribosomes lacking RACK1/Asc1 are deficient in the translation of short mRNAs and have an increased association with certain autophagy-related mRNAs (Thompson et al 2016;Kim et al 2017). Furthermore, both cap-dependent and IRES-mediated translations are reduced in RACK1-depleted cells (Majzoub et al 2014;Gallo et al 2018).…”

Section: Ribosomal Protein Contentmentioning

confidence: 99%

Does functional specialization of ribosomes really exist?

Ferretti

Karbstein

2019

RNA

105

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It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gainof-function assays and the demonstration of inducibility under physiological conditions.

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RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy

Cited by 24 publications

References 58 publications

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs CytochromecOxidase Subunit Expression inLeishmania major

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs CytochromecOxidase Subunit Expression inLeishmania major

eIF 5A is required for autophagy by mediating ATG 3 translation

Does functional specialization of ribosomes really exist?

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