LC3 and GABARAP independent autophagy of misfolded procollagen in mouse osteoblasts

Bone synthesis should depend on autophagy because over 10% of type I procollagen (PC1) - a heterotrimer of COL1A1 and COL1A2 chains and the precursor of the main bone matrix molecule - is misfolded and rerouted from osteoblast endoplasmic reticulum (ER) to lysosomes. However, osteoblast-specific macroautophagy knockouts in mice have produced only mild bone effects. To reconcile these observations, we compared how hypomorphic expression and a conditional knockout (cKO) of <i>Atg5</i> - encoding a protein required for autophagosome formation - affected <i>Col1a2</i>^G610C/+ versus wild-type <i>Col1a2</i>^+/+ osteoblasts <i>in vivo</i> and <i>in vitro</i>. The Gly610-to-Cys substitution (G610C) in the triple helical region of the COL1A2/proα2(I) chain increases PC1 misfolding, causing its accumulation in the ER, cell stress, and osteoblast malfunction. Because autophagy reroutes misfolded PC1 from the ER to lysosomes, disruption of PC1 autophagy should significantly increase osteoblast malfunction and bone pathology in <i>Col1a2</i>^G610C/+ mice. Nonetheless, the present study revealed only minor effects of the <i>atg5</i> cKO on osteoblast function and bone formation in the <i>Col1a2</i>^G610C/+ mice, like in <i>Col1a2</i>^+/+ controls. The cKO did not reduce the autophagy flux of misfolded G610C or wild-type PC1 in primary osteoblast cultures, even though the LC3 and GABARAP lipidation and therefore autophagosome formation were disrupted. Live-cell imaging in <i>atg5</i> cKO osteoblasts demonstrated that PC1 was efficiently delivered to lysosomes without LC3 via ER exit site (ERES) microautophagy. Taken together, these observations indicate that LC3- and GABARAP-independent ERES microautophagy is the primary pathway of misfolded procollagen degradation in osteoblasts both in culture and <i>in vivo</i>.<b>Abbreviations</b>: ATG5: autophagy related 5; ATG7: autophagy related 7; Baf: bafilomycin A₁; BFA: brefeldin A; BGLAP/Ocn/osteocalcin: bone gamma-carboxyglutamate protein; COL1A1/proα1(I): collagen type I alpha 1 chain; COL1A2/proα2(I): collagen type I alpha 2 chain; cKO: conditional knockout; ER: endoplasmic reticulum; ERES: ER exit site; G610C mutation: COL1A2 p.Gly706Cys replacing Gly in position 610 from the start of the triple helix with Cys; GABARAP: GABA type A receptor-associated protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAR: mineral apposition rate; Ob: osteoblast; Oc: osteoclast; OI: osteogenesis imperfecta; PC1: procollagen type I, a heterotrimer of two COL1A1 and one COL1A2 chains, precursor of collagen type I; PDI: protein disulfide isomerase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SP7/osterix: Sp7 transcription factor; SQSTM1/p62: sequestosome 1; WT: wild type.