Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.
Bibliographical noteFunding Information:
We thank W.S. Hancock (Boston, MA), N.-O. Ku (Seoul), P.M. Lackie (Southampton), Jon Sonderholm (Seoul), D.J. Taatjes (Burlington, VT) and G.H.F. Yam (Hong Kong) for critical reading of the manuscript. We are grateful to Tamara Locher and Roger Santimaria for skillful technical assistance. Insook Jang is recipient of a fellowship from the Brain Korea 21 program. Funding was received by the Swiss National Science Foundation (to J.R.), the Canton of Zurich (to J.R.), by the World Class University Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-2008-000-10086-0) (to J.R. and J.W.C), the National Research Foundation of Korea by the Ministry of Education, Science and Technology (2010-0027736) (to J.R. and J.W.C) and partly by the National Research Foundation funded by the Korean Government (2012R1A2A1A05026333) (to J.W.C.).
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Molecular Biology
- Cellular and Molecular Neuroscience
- Cell Biology