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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Biol Chem. 2008 Aug;389(8):1107–1115. doi: 10.1515/BC.2008.122

Figure 4. Substrate-gating mutants of GlpG display increased activity in mammalian cells.

Figure 4

Western analysis of GlpG expression and activity against Spitz in transfected COS cells. The top panel shows accumulation of cleaved GFP-Spitz as a secreted product in cell culture media, as detected by anti-GFP. Note that while wildtype GlpG did not produce a cleaved GFP-Spitz product above background, the cleaved product can be detected in the media fraction with all three GlpG mutants. The positive control, Drosophila rhomboid-1 (DmR1) displayed strong activity against Spitz. The middle panel represents GFP-Spitz in transfected cells, with the upper band being uncleaved GFP-Spitz, and the lower band being cleaved GFP-Spitz prior to being secreted. The arrow indicates the barely detectable GFP-Spitz cleavage band produced by wildtype GlpG. Note that all three GlpG mutants increase the amount of cleaved Spitz in the cell lysates. The lower panel depicts expression levels of each rhomboid, all of which were tagged with a triple hemagglutinin tag and detected by anti-HA western. Note that only 1/4 of the DmR1 cell lysate volume was loaded compared to the GlpG lysates since its expression was already so much greater than that of GlpG proteins. The W157A+F232A GlpG mutant was expressed at a higher level than other GlpG proteins. The location of protein mass standards (in kDa) are depicted on the left.