Abstract
α1-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-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.—Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.
Highlights
Cells were permeabilized on ice for 5 min with 40 mg/ml digitonin diluted in complete transport buffer (CTB; TB with protease inhibitor cocktail [Roche, Basel, Switzerland] and 1 mM DTT), washed twice with CTB, and incubated in CTB thereafter
To visualize a1-antitrypsin trafficking in live cells, we fused wild-type M- or polymerogenic Z-a1-antitrypsin to an endoplasmic reticulum (ER)-targeted yellow fluorescent protein (YFP) separated by a flexible (Gly4Ser)3 linker
Cells were cotransfected with the tagged-a1-antitrypsin and with a marker of the ER or the Golgi apparatus (Gmx33-green fluorescent protein (GFP)) [19] and imaged by confocal microscopy
Summary
The inclusion bodies of polymerized a1-antitrypsin contain the endoplasmic reticulum (ER)-resident chaperones BiP and PDI, and are frequently decorated with ribosomes [6, 7] These inclusions appear to differ from healthy ER in other respects; for example, they have been reported to lack the chaperone calnexin (CNX) and have wide lumens of .500 nm compared to ,100 nm for normal ER [7, 8]. We and others have reported that polymerization of a1-antitrypsin within the ER leads to an exaggerated unfolded protein response if ER stress is caused by other means [8, 12] We showed that this correlates with reduced mobility of small ER marker proteins in cells containing inclusions [8]. Small proteins rapidly exchange between physically distinct inclusion bodies by vesicular transport that requires cytosol, is sensitive to N-ethylmaleimide (NEM) and dependent on Sar and sec22B, but is independent of atlastin-mediated homotypic fusion of the ER
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