Abstract
Endoplasmic reticulum (ER) α-1, 2-mannosidase (ERManI) contributes to ER-associated protein degradation (ERAD) by initiating the formation of degradation signals on misfolded N-linked glycoproteins. Despite its inferred intracellular location, we recently discovered that the mammalian homologue is actually localized to the Golgi complex. In the present study, the functional role of Golgi-situated ERManI was investigated. Mass spectrometry analysis and coimmunoprecipitation (co-IP) identified a direct interaction between ERManI and γ-COP, the gamma subunit of coat protein complex I (COPI) that is responsible for Golgi-to-ER retrograde cargo transport. The functional relationship was validated by the requirement of both ERManI and γ-COP to support efficient intracellular clearance of the classical ERAD substrate, null Hong Kong (NHK). In addition, site-directed mutagenesis of suspected γ-COP-binding motifs in the cytoplasmic tail of ERManI was sufficient to disrupt the physical interaction and ablate NHK degradation. Moreover, a physical interaction between NHK, ERManI, and γ-COP was identified by co-IP and Western blotting. RNA interference-mediated knockdown of γ-COP enhanced the association between ERManI and NHK, while diminishing the efficiency of ERAD. Based on these findings, a model is proposed in which ERManI and γ-COP contribute to a Golgi-based quality control module that facilitates the retrieval of captured ERAD substrates back to the ER.