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1.
Life Sci Alliance ; 5(7)2022 03.
Article in English | MEDLINE | ID: mdl-35314489

ABSTRACT

Retrograde protein transport from the cell surface and endosomes to the TGN is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. We used a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN for the cation-dependent MPR (CDMPR) with and without rapid or gradual inactivation of candidate machinery proteins. Although knockdown of retromer (Vps26), epsinR, or Rab9a reduced CDMPR arrival to the TGN, no effect was observed upon silencing of TIP47. Strikingly, when retrograde transport was analyzed by rapamycin-induced rapid depletion (knocksideways) or long-term depletion by knockdown of the clathrin adaptor AP-1 or of the GGA machinery, distinct phenotypes in sulfation kinetics were observed, suggesting a potential role of GGA adaptors in retrograde and anterograde transport. Our study illustrates the usefulness of derivatized, sulfation-competent nanobodies, reveals novel insights into CDMPR trafficking biology, and further outlines that the selection of machinery inactivation is critical for phenotype analysis.


Subject(s)
Single-Domain Antibodies , trans-Golgi Network , Cations , Endosomes/metabolism , HeLa Cells , Humans , Single-Domain Antibodies/metabolism , trans-Golgi Network/metabolism
2.
Elife ; 92020 07 13.
Article in English | MEDLINE | ID: mdl-32657755

ABSTRACT

A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.


Subject(s)
Fungal Proteins/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae/physiology , Cytosol/metabolism , Green Fluorescent Proteins/metabolism , Mitochondria/physiology , Protein Transport
3.
Methods ; 113: 91-104, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27725303

ABSTRACT

By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Cell Nucleus/enzymology , Cytosol/enzymology , Mitochondria/enzymology , Ribosomes/enzymology , Saccharomyces cerevisiae/enzymology , Amino Acyl-tRNA Synthetases/classification , Amino Acyl-tRNA Synthetases/genetics , Antibodies/chemistry , Blotting, Western/methods , Cell Compartmentation , Cell Fractionation/methods , Cell Line , Cell Nucleus/ultrastructure , Cytosol/ultrastructure , Fluorescent Antibody Technique/methods , Gene Expression , Humans , Mitochondria/ultrastructure , Protein Transport , Ribosomes/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure
4.
Mol Cell ; 56(6): 763-76, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25453761

ABSTRACT

In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control.


Subject(s)
Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/genetics , Cell Nucleus/genetics , Gene Expression , Gene Expression Regulation, Fungal , Mitochondria/genetics , Multienzyme Complexes , Protein Multimerization , Proton-Translocating ATPases/metabolism , RNA-Binding Proteins/physiology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/physiology
5.
FEBS Lett ; 588(23): 4268-78, 2014 Nov 28.
Article in English | MEDLINE | ID: mdl-25315413

ABSTRACT

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and ancient enzymes, mostly known for their essential role in generating aminoacylated tRNAs. During the last two decades, many aaRSs have been found to perform additional and equally crucial tasks outside translation. In metazoans, aaRSs have been shown to assemble, together with non-enzymatic assembly proteins called aaRSs-interacting multifunctional proteins (AIMPs), into so-called multi-synthetase complexes (MSCs). Metazoan MSCs are dynamic particles able to specifically release some of their constituents in response to a given stimulus. Upon their release from MSCs, aaRSs can reach other subcellular compartments, where they often participate to cellular processes that do not exploit their primary function of synthesizing aminoacyl-tRNAs. The dynamics of MSCs and the expansion of the aaRSs functional repertoire are features that are so far thought to be restricted to higher and multicellular eukaryotes. However, much can be learnt about how MSCs are assembled and function from apparently 'simple' organisms. Here we provide an overview on the diversity of these MSCs, their composition, mode of assembly and the functions that their constituents, namely aaRSs and AIMPs, exert in unicellular organisms.


Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/metabolism , Evolution, Molecular , Protein Structure, Quaternary , Animals , Humans , Protein Structure, Tertiary , Species Specificity
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