Folding proteome of Yarrowia lipolytica targeting with uracil permease mutants.
The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.
INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France. dominique.swennen@grignon.inra.fr
Dominique
D
Swennen
Céline
C
Henry
Jean-Marie
JM
Beckerich
D000595:Amino Acid Sequence; D000963:Antimetabolites / Q000378:metabolism / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D025141:Drug Resistance, Fungal / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D033704:Nucleotide Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism
D000963:Antimetabolites; C035528:FUR4 protein, S cerevisiae; D005656:Fungal Proteins; D008565:Membrane Proteins; D033704:Nucleotide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D005472:Fluorouracil