Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling

Schmitt, Kerstin; Smolinski, Nadine; Neumann, Piotr; Schmaul, Samantha; Hofer-Pretz, Verena; Braus, Gerhard H.; Valerius, Oliver

doi:10.1128/mcb.00279-16

Cited by 32 publications

(59 citation statements)

References 73 publications

(82 reference statements)

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“…Peptides were desalted over an Empore Octadecyl C18 47-mm extraction disks 2215 (Supelco) according to Rappsilber et al (2007), dried, and dissolved in a 20 μL 0.1% formic acid. Then, the peptides were subjected to LC-MS/MS (Schmitt et al, 2017). In detail, nano-flow liquid chromatography was done with the RSLCnano Ultimate 3000 (Thermo Fisher Scientific) system.…”

Section: Proteomics Sample Preparation and Lc/ms Analysis Of Peptidesmentioning

confidence: 99%

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins

et al. 2018

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The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.

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Section: Proteomics Sample Preparation and Lc/ms Analysis Of Peptidesmentioning

confidence: 99%

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins

et al. 2018

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“…S1A,B). Third, RACK1 has extraribosomal functions in several cell lines that might be linked to transformation or only operate in specific cell types or contexts (Gallo and Manfrini, 2015;Schmitt et al, 2017). By contrast, several cell typesincluding normal cells, such as NHDFsdegrade extra-ribosomal RACK1 and restrict its function to the ribosome (Ceci et al, 2003;Gallo et al, 2018;Gerbasi et al, 2004;Jha et al, 2017;Johnson et al, 2019;Romano et al, 2019;Sengupta et al, 2004).…”

Section: The Loop Region Controls Rack1 Interactions With Eif6mentioning

confidence: 99%

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity in a loop domain

Rollins

Jha

Bartom

et al. 2019

Journal of Cell Science

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Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved β-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plantbut not uncharged yeast or human loopenhances translation of mRNAs with adenosine-rich 5′ untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.

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“…The Coomassie blue-stained protein bands were then excised and processed as described by Shevchenko et al (1996). Peptides were desalted over a Supelco C18 column (Sigma-Aldrich) according to Rappsilber et al (2007) and then subjected to LC-MS/MS (Schmidtt et al, 2017). Protein abundance was quantified using iBAQ label-free quantification implemented in MaxQuant software (Schaab et al, 2012) and the values were calculated as a percentage of all values in one sample.…”

Section: Ld Isolations and Intensity-based Quantification (Ibaq) Labementioning

confidence: 99%

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

et al. 2017

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Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady-state maintenance and turnover of plant LDs, particularly in non-seed tissues, are relatively unknown. Previously, we showed that the LD-associated proteins (LDAPs) are a family of plant-specific, LD surface-associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two-hybrid library using the Arabidopsis LDAP3 isoform as 'bait' in an effort to identify other novel LD protein constituents. One of the candidate LDAP3-interacting proteins was Arabidopsis At5g16550, which is a plant-specific protein of unknown function that we termed LDIP (LDAP-interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α-helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T-DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds.

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Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling

Cited by 32 publications

References 73 publications

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity in a loop domain

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

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