Membrane Topology of the Yeast Endoplasmic Reticulum-localized Ubiquitin Ligase Doa10 and Comparison with Its Human Ortholog TEB4 (MARCH-VI)

Abstract

Quality control machinery in the endoplasmic reticulum (ER) helps ensure that only properly folded and assembled proteins accumulate in the ER or continue along the secretory pathway. Aberrant proteins are retrotranslocated to the cytosol and degraded by the proteasome, a process called ER-associated degradation. Doa10, a transmembrane protein of the ER/nuclear envelope, is one of the primary ubiquitin ligases (E3s) participating in ER-associated degradation in Saccharomyces cerevisiae. Here we report the membrane organization of the 1319-residue Doa10 polypeptide. The topology was determined by fusing a dual-topology reporter after 16 different Doa10 fragments. Our results indicate that Doa10 contains 14 transmembrane helices (TMs). Based on protease digestion of yeast microsomes, both the N-terminal RING-CH domain and the C terminus face the cytosol. Notably, the experimentally derived topology was not predicted correctly by any of the generally available TM prediction algorithms. Bioinformatic analysis and in silico mutagenesis guided the topological studies through problematic regions. The conserved TD domain in Doa10 includes three TMs. These TMs might function in cofactor binding or substrate recognition, or they might be part of a retrotranslocation channel. The Derlins were previously proposed to provide such channels, but we show that the two yeast Derlins are not required for degradation of Doa10 membrane substrates, as was found before for the Sec61 translocon. Finally, we provide evidence that the likely human Doa10 ortholog, TEB4 (MARCH-VI), adopts a topology similar to that of Doa10.In eukaryotes, most proteins that must traverse or insert into a lipid bilayer are first translocated from the cytoplasm into or through the ER 2 membrane, after which they are sorted to various cellular compartments. A resident quality control machinery surveys the ER and eliminates misfolded, unassembled, or aberrantly modified proteins by the ERAD system (for review, see Refs. 1-3). In addition to its role in quality control, ERAD is also employed in the physiologically regulated degradation of normal ER proteins, such as the rate-limiting enzyme for sterol biosynthesis, hydroxymethylglutaryl-coenzyme A reductase (4). Activity of the ERAD system can sometimes also have deleterious effects, as in cystic fibrosis in humans. The most common mutation causing this disease is a single amino acid deletion (⌬F508) in the cystic fibrosis conductance regulator protein, which triggers the rapid proteasomedependent degradation of the protein at the ER (5).ERAD substrates are retrotranslocated from the ER lumen or ER membrane to the cytosol before or concomitant with degradation by the 26 S proteasome (6, 7). Ubiquitination of the substrate is generally required for both efficient retrotranslocation and degradation by the proteasome (3). Ubiquitination involves the conjugation of ubiquitin polymers to a substrate protein (8), which is accomplished through a linked series of enzymatic reactions catalyzed by three classes of enzyme...

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

“…The previously proposed function of the ER membrane-embedded ligases Hrd1/Der3 and Doa10 as possible channel components for substrate retrotranslocation (9,16,46) cannot hold true for Ubr1-triggered ERAD of the tested substrates in the absence of the canonical ligases. The mechanism underlying the retrotranslocation of proteins from the ER to the cytoplasm in the absence of the canonical ligases using the Ubr1-dependent ERAD route will be a challenging goal in the future.…”

“…It has been known for years that two polytopic ligases located in the ER membrane, Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), play a central role in ERAD in yeast, by directing ERAD substrates to proteasomal degradation (11)(12)(13)(14)(15)(16). Previous studies on a variety of ERAD substrates revealed that, in most cases, the absence of one of the two canonical ER ubiquitin ligases does not lead to a complete block of degradation of the tested substrate.…”

Analysis of Prelamin A Biogenesis Reveals the Nucleus to be a CaaX Processing Compartment

“…It was recently shown that a notable exception to this paradigm is the yeast E3 ubiquitin ligase, Doa10p. Doa10p was long known to reside in the ER membrane (Swanson et al, 2001;Kreft et al, 2006). However, recent data indicate that Doa10p exhibits dual steady-state localizations, residing and functioning in both the ER membrane where it mediates ER-associated degradation of membrane and secretory proteins, and in the INM where it mediates degradation of the nuclear transcription factor MAT␣2 (Deng and Hochstrasser, 2006).…”

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

“…The 1319-residue Doa10 E3 ligase has 14 transmembrane segments, a topology that appears to be conserved in the mammalian ortholog MARCH6 (TEB4) (11). Both the N and C termini of Doa10/MARCH6 are exposed to the cytosol with ubiquitin ligase activity derived from an N-terminal RING-CH domain, a subclass of RING domains (12).…”