Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system.
Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-alpha factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61alpha. In addition, pro-alpha factor photocrosslinked derlin-1, but not Sec61alpha. Thus, derlin-1 appears to be involved in pro-alpha factor retrotranslocation.
Wahlman
Judit
J
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
DeMartino
George N
GN
Skach
William R
WR
Bulleid
Neil J
NJ
Brodsky
Jeffrey L
JL
Johnson
Arthur E
AE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006031:Glycosylation; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D046188:Protein Modification, Translational; D011498:Protein Precursors / Q000378:metabolism*; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
C487709:DERL1 protein, human; D005456:Fluorescent Dyes; C048632:MF(ALPHA)1 protein, S cerevisiae; D008565:Membrane Proteins; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases