Exploring the molecular factors governing the unconventional protein secretory pathway using quantitative proteomics

To ensure tissue homeostasis, eukaryotic cells secrete factors involved in various biological processes such as defense against foreign pathogens, extracellular matrix remodeling, damaged cells etc. While most proteins are exported to the extracellular space via the classical secretory pathway (ER-Golgi route), an increasing category of proteins are being released independent of this pathway, via the unconventional secretory pathway (UPS). UPS cargoes are proteins with apparently unrelated functions and include growth cytokines (IL-1β, IL-1α, IL-18), neuropeptides (ACBP), redox enzymes (SOD1), factors (FGF2), ion channels (CFTR) and in general, few common factors have been identified between the molecular processes involved in their release. GRASP55 (Golgi-reassembling stacking proteins 55), has been linked with the UPS of several such cargoes and alterations in intracellular GRASP55 level results in perturbed UPS. Amongst these, the secretion of the pro-inflammatory cytokine IL-1β was shown to be GRASP55-dependent from primary macrophages under physiologically relevant conditions.

We used a GRASP55 knock-out (G55-/-) mouse strain to underpin the molecular mechanisms involved in unconventional protein secretion from primary Mouse Embryonic Fibroblasts (MEFs) subjected to tunicamycin treatment, an N-glycosylation inhibitor which results in endoplasmic reticulum (ER) stress. We undertook a quantitative proteomics approach to obtain a global picture of the proteome relative changes from both WT and G55-/- MEF cells by combining offline high pH peptide fractionation with LC-MS/MS analysis to discover novel factors participating in the unconventional secretory pathway. We characterized the proteome of the primary cell cultures up to a depth of over 5000 proteins. Ablation of GRASP55 resulted in alterations of molecular factors involved in protein degradation, including the modification of the protein expression level of some ER and Golgi resident factors such as Emc4 or Lman2, involved in the sorting of the glycoproteins from the early secretory pathway. Moreover, we observed that during ER stress treatment BiP, Dnajc3 and the molecular chaperon Hsp90b1 are up-regulated compared with the control cells for both the WT and G55-/- primary cell lines. In general, tunicamycin treatment resulted in the modification of the relative expression level of proteins from the secretory pathway involved in various cell to cell communication signaling. Our results provide a detailed image of the GRASP55 dependent molecular effectors from the unconventional secretory pathway, with a potential-role of molecular hubs in the GRASP55-dependent secretome.