Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD.
The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.
Taxis
Christof
C
Institut für Biochemie, Universität Stuttgart, 70569 Stuttgart, Germany.
Hitt
Reiner
R
Park
Sae-Hun
SH
Deak
Peter M
PM
Kostova
Zlatka
Z
Wolf
Dieter H
DH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
C098515:DER1 protein, S cerevisiae; C494647:DNAJB1 protein, human; D005656:Fungal Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011500:Protein Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C070319:YDJ1 protein, S cerevisiae; C070972:HsP104 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D003513:Cycloheximide