Distinct machinery is required for the ER-associated degradation of a multispanning membrane protein and a soluble lumenal protein.
Gregory Huyer (1), Wachirapon F. Piluek (1), Jeffrey L. Brodsky (2), Susan Michaelis (1)
(1) Cell Biology, Johns Hopkins Medical School, 725 N. Wolfe Street, Baltimore, MD, 21205, USA;
(2) Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane is monitored by ER quality control (ERQC). Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. While lumenal and integral membrane ERAD substrates both require the proteasome for their degradation, the ERQC machinery for these two classes of proteins likely differs because of their distinct topologies. Here, we establish the requirements for the ERAD of a multispanning membrane protein with a cytosolic mutation (Ste6p*), and compare them with those for a mutant lumenal protein (CPY*). We show that turnover of Ste6p*, unlike CPY*, is dependent on the E3 ubiquitin ligase Doa10p. Furthermore, cytosolic chaperones and co-chaperones are important in ERAD of Ste6p*, while the ER lumenal chaperone Kar2p is dispensable, opposite to their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to involve the Sec61p translocon pore. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of multispanning membrane proteins with a cytosolic mutation employs distinct machinery from that of soluble lumenal substrates. These studies indicate that Ste6p* is a valuable model substrate to dissect ERAD machinery.
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