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. 2019 Dec 5;61(2):229–243. doi: 10.1194/jlr.RA119000431

Fig. 6.

Fig. 6.

Sre1 cleavage dependency on STP1. A: Western blot assay of protein extracts obtained from X. dendrorhous strains carrying Sre1 or Sre1N labeled with 3xFLAG at its N-terminus after 48 h of culture in YM medium with constant agitation. Strains CBS 6938, CBS.sre1−, and CBS.Δstp1, without FLAG-tagged Sre1/Sre1N proteins, were included as controls (lanes 1, 2, and 3, respectively). Lanes 4–9 correspond to protein extracts from FLAG-Sre1- or FLAG-Sre1N-labeled strains: CBS.FLAG.SRE1 (lane 4), CBS.FLAG.SRE1.Δstp1 (lane 5), CBS.Δstp1.FLAG.SRE1N (lane 6), CBS.FLAG.SRE1N (lane 7), CBS.cyp61−.FLAG.SRE1 (lane 8), and CBS.cyp61−.FLAG.SRE1.Δstp1 (lane 9). Anti-ubiquitin was included as a control. FLAG-Sre1 (∼130 kDa) and FLAG-Sre1N (∼95 kDa) bands are indicated. M, PageRuler Plus prestained protein ladder (Thermo Fisher Scientific Inc.). B: Amino acid sequence alignment of the first TM segment of H. sapiens SREBP-2 [GenBank: Q12772.2] and the predicted first TM segment of Sre1 from C. neoformans [GenBank: XP_012052219.1] (CnSre1) and X. dendrorhous [GenBank: MK368598] (XdSre1). The first TM segment predicted in each protease is shown in blue. The arrow in SREBP-2 indicates the S2P cleavage site between a leucine and a cysteine residue (53), and asterisks in CnSre1 indicate the end of two truncated constructions of CnSre1 (residues 501 and 535), which suggests that Sre1 is cleaved by CnStp1 at the first TM segment (21). Potential helix-destabilizing residues are highlighted in yellow.