1. Requirements for Signal Peptide Peptidase-Catalyzed Intramembrane Proteolysis 
Marius K Lemberg, Bruno Martoglio 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)00655-X

2. Figure 1 Analyzing Processing of Signal Peptides
(A) Signal sequences of bovine prolactin (Prl), human MHC class I molecule HLA-A*0301, rat calreticulin (CRT), human RNase A, vesicular stomatitis virus G protein (VSV-G), and human cytomegalovirus glycoprotein UL40 (gpUL40). h regions are underlined; arrows indicate the signal peptidase (SPase) cleavage site; bows indicate the approximate SPP cleavage site in the Prl and HLA-A*0301 signal peptide.
(B) Processing of signal peptides in microsomes. In vitro translation of mRNA coding for the signal sequence plus 100 residues (p-xx/100) of the indicated proteins in the absence (lanes 1) or in the presence of ER-derived microsomes (lanes 2 and 3) and SPP inhibitor (Z-LL)2-ketone (lanes 3). Microsomes were isolated and analyzed by SDS-PAGE, and radiolabeled proteins visualized by phosphorimaging. Note that the extent of radiolabeling of signal peptide (SP) and cognate translocated protein (xx/100) depends on the content of radiolabeled amino acids and, therefore, varies from protein to protein. Lanes 4, in vitro-translated reference signal peptide.
(C) Quantification of signal peptide processing. Bars indicate the relative amounts of signal peptide obtained in lane 2 compared to the corresponding lane 3, where SPP was inhibited (set to 0% processing). Each value was calculated from three independent experiments and corrected for variation of translocation.
Molecular Cell  , DOI: ( /S (02)00655-X)

3. Figure 2 Influence of Charged Residues Flanking the h Region
(A) Sequences of signal peptide mutants HCMV gpUL40Δ2-18, HCMV gpUL40T31T33, PrlR25, and PrlR25R27. Exchanged residues are highlighted in bold; h regions are underlined.
(B) Quantification of signal peptide processing. Processing of signal peptides was investigated in microsomes and quantified as described in Figure 1.
Molecular Cell  , DOI: ( /S (02)00655-X)

4. Figure 3 Effect of Helix-Breaking Residues in the h Region
(A) Sequences of signal peptide mutants RNase ASN16, PrlLL21, PrlGL21, and PrlLL21R25. Exchanged residues are highlighted in bold; h regions are underlined.
(B) Quantification of signal peptide processing. Processing of signal peptides was investigated in microsomes and quantified as described in Figure 1.
Molecular Cell  , DOI: ( /S (02)00655-X)

5. Figure 4
Requirements for Signal Peptide Processing Investigated in Live Cells
(A) Signal sequences of wild-type HCV core (HCV Cwt) and signal peptide mutants (HCV CV180 and HCV CLV184). h regions are underlined; exchanged residues are highlighted in bold.
(B) Expression of HCV CE1E2 polyprotein of wild-type and signal peptide mutants and analysis of core protein by Western blotting. Extracts from BHK cells expressing wild-type and mutant HCV CV180 were resolved on Tris-glycine acrylamide gels (lanes 3). To resolve proteins of mutant HCV CLV184, samples were analyzed on a Tris-bicine gel, where the 191 residue long peptide showed greater mobility than the shorter 179 residue long peptide (lane 3, bottom panel). Lanes 1, 2, and 4 show in vitro-translated reference peptides corresponding to the N-terminal 179, 182, and 191 residues of the HCV polyprotein.
(C) Quantification of processing. The relative amounts of processed core protein (179 residues long) were correlated with the total amount of core species (processed + nonprocessed 191 residue long protein = 100%).
(D) Immunfluorescence of core protein combined with staining of lipid droplets. Scale bar, 5 μm.
Molecular Cell  , DOI: ( /S (02)00655-X)

6. Figure 5
Signal Peptide Processing in Intact Membranes Requires Preceding Cleavage by Signal Peptidase
(A) Signal sequence of mutant PrlPP29. Exchanged residues are highlighted in bold; h region is underlined; crossed arrow indicates position of signal peptidase (SPase) cleavage site in the wild-type protein.
(B) Mutations in PrlPP29 abolish cleavage by signal peptidase in intact membranes. mRNA coding for p-Prl/130 and p-PrlPP29/130 was translated in vitro in the absence (lanes 1 and 5) or in the presence of ER-derived microsomes (lanes 2–4 and 6–8). Membranes were isolated and treated with proteinase K (Prot. K; lanes 3 and 7) or proteinase K plus Triton X-100 (TX100; lanes 4 and 8).
(C) Mutations in PrlPP29 do not affect cleavage by detergent solubilized SPP activity. In vitro-translated signal peptides of Prl and PrlPP29 (lanes 1 and 4) were incubated with detergent-solubilized ER membrane proteins containing SPP in the absence (lanes 2 and 5) or in the presence of SPP inhibitor (Z-LL)2-ketone (lanes 3 and 6).
Molecular Cell  , DOI: ( /S (02)00655-X)

7. Figure 6
Intramembrane Proteolysis by SPP in Live Cells Requires Preceding Cleavage by Signal Peptidase
(A) Signal sequence of mutant HCV CspkoE1. Exchanged residues are highlighted in bold; h region is underlined; crossed arrow indicates position of signal peptidase (SPase) cleavage site in the wild-type protein.
(B) Mutations in HCV CspkoE1 abolish cleavage by signal peptidase in microsomes. Synthesis of HCV CspkoE1/100 by in vitro translation in the absence (lane 1) or in the presence of ER-derived microsomes (lanes 2–4), acceptor tripeptide to inhibit N-glycosylation (lanes 3 and 4), and (Z-LL)2-ketone (lane 4). Dots indicate glycosylated CE1/100.
(C) Mutations in HCV Cspko abolish cleavage by signal peptidase but not SPP. mRNAs coding for HCV CE1/4 (lanes 1–3) and HCV CspkoE1/4 (lanes 4–6) were translated in vitro in the absence (lanes 1 and 4) or in the presence of ER-derived microsomes (lane 2, 3, 5, and 6) and (Z-LL)2-ketone (lanes 3 and 6). Lanes 7 and 8 show in vitro-translated reference peptides corresponding to the N-terminal 179 and 191 residues of corespko (asterisks).
(D) Mutations in HCV Cspko abolish cleavage by signal peptidase in live cells. HCV CE1E2, HCV CspkoE1E2, HCV CE1/4, and HCV CspkoE1/4 were expressed in BHK cells, and cell extracts were probed by Western blot analysis with core-specific antibody (left panel), and E1-specific antibody before or after treatment with endoglycosidase H (Endo H; right panel). Dots indicate nonprocessed, glycosylated CspkoE1.
(E) Immunfluorescence of core protein combined with staining of lipid droplets of cells expressing HCV CspkoE1E2, HCV CspkoE1/4, and HCV CLV184E1/4 (see also Figure 4). Scale bar, 5 μm.
Molecular Cell  , DOI: ( /S (02)00655-X)