Misfolded proteins transiting through the secretory pathway are translocated from the endoplasmic reticulum (ER) to the cytosol, where they are degraded via ER-associated degradation (ERAD). In Saccharomyces cerevisiae, this involves polyubiquitylation by the ER membrane-associated E3 ligases Hrd1 and Doa10, although loss of both proteins does not completely block degradation. This study reveals that the cytoplasmic E3 ligase Ubr1 is also involved in ERAD. Ubr1 ubiquitylated Ste6* (a truncated version of pheromone mating type A transporter), promoting its proteasomal degradation, in the absence of both Hrd1 and Doa10, or under heat shock or ethanol treatment. The chaperone Ssa1 and the AAA+ ATPase Cdc48 were required for this process. Notably, Ubr1 was integral for the ubiquitylation of CTCF (a human protein that is completely degraded by ERAD when expressed in yeast) under standard growth conditions and in the presence of Hrd1 and Doa10.