Introduction and objectives Alpha-1-antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-alpha-1-antitrypsin, which has a propensity to self-associate into polymers that become entrapped within inclusions of endoplasmic reticulum (ER). This predisposes to the development of cirrhosis, while the resulting paucity of circulating alpha-1-antitrypsin leads to early-onset emphysema. It is unclear whether intracellular inclusions are physically or functionally connected to the main ER network in Z-alpha-1-antitrypsin expressing cells. In this study, we sought to clarify the behaviour of proteins within inclusion bodies to further our understanding of the consequences of Z-alpha-1-antitrypsin expression on cellular dysfunction.
Methods We created YFP tagged wild-type and Z-alpha-1-antitrypsin constructs and expressed them in a cell model along with other fluorescently tagged proteins of interest. Using live-cell imaging including photobleaching techniques, we assessed the relative mobilities of alpha-1-antitrypsin and other ER resident proteins. We further assessed the nature of inter-inclusion protein trafficking by creating a permeabilised cell system in which the cytosol could be manipulated or removed.
Results We have shown that inclusions are translationally active ER fragments in which polymerisation occurs. Using fluorescence recovery after photobleaching (FRAP), we observed that despite inclusions containing immobile polymeric alpha-1-antitrypsin, small ER resident proteins including the ER chaperone BiP are able to diffuse freely within them (Figure 1). We observed that inclusions are physically separated from the tubular ER network but despite this, cargo is transported between inclusions in a cytosol-dependent fashion that is dependent on vesicular trafficking components and may involve the ER-Golgi intermediate compartment (ERGIC).
Conclusions We propose that protein movement between physically separated ER inclusions via ER-ERGIC recycling acts to minimise ER heterogeneity in Z-alpha-1-antitrypsin expressing cells. This may reduce the toxic effects of polymer accumulation via increased availability of, for example, ER chaperones within inclusions and is consistent with in vivo observations of a striking lack of toxicity in cells expressing Z-alpha-1-antitrypsin.
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