Journal: The EMBO Journal
Article Title: Bacterial rhomboid proteases mediate quality control of orphan membrane proteins
Figure Lengend Snippet: Rhomboid cleavage licenses further degradation of substrates Western blot analysis (probing with an anti‐V5 mAb) to detect degradation of N‐terminally V5‐tagged wild‐type (WT) or modified (P 300 A) HybA in S. sonnei Δ rhom7 chromosomally expressing wild‐type (+) or inactive (S 201 A) GlpG with (+) or without (−) HybB. Degradation of V5‐tagged HybA at times after blocking protein translation at T 0 in the presence (+) or absence (−) of HybB. Western blot analysis of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdoH in S. sonnei Δ rhom7 chromosomally expressing wild‐type (+) or inactive (S 201 A) GlpG with (+)/without (−) FdoI. Western blot analysis of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdnH in S. sonnei Δ rhom7 with wild‐type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (−) FdnI. Degradation of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdoH (E) or FdnH (F) in S. sonnei Δ rhom7 with wild‐type (+) or inactive (S 201 A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI or FdnI (−), respectively, +/− exposure to 400 μM CuCl 2 for 30 min. Data information: Rhomboid substrates that are uncleaved, cleaved by GlpG or cleaved by Rhom7 are marked by black, red and blue arrows, respectively. Degradation products post‐rhomboid cleavage are marked by green arrows. RecA, loading control. Source data are available online for this figure.
Article Snippet: Specific point mutations or addition of a sequence encoding a 3xHA tag was introduced by Gibson cloning (New England Biolabs) with appropriate primers. pUC19‐based vectors (Yanisch‐Perron et al , ) were constructed by assembling appropriate glpG fragments into Sph I/Hind III‐linearised pUC19 by NEBuilder, and Gibson cloning was used to introduce specific point mutations or sequences encoding a triple‐HA tag. pLAC101 is a low‐copy‐number plasmid for expressing proteins under IPTG induction. pLAC101 contains the pSC101 origin (amplified with pGL1315/pGL1316 from pUA139) (Zaslaver et al , ), the kanamycin resistance gene and lacI with a cloning site amplified from pNCC1 (Wormann et al , ) with primers pGL897/pGL1314 and pGL894/pGL1314, respectively. pLAC101V5‐hybA /V5‐hybA P300A were generated by assembling appropriate fragments into Pac I/Kpn I‐linearised pLAC101.
Techniques: Western Blot, Modification, Expressing, Blocking Assay, Plasmid Preparation