1. Protein Folding during Cotranslational Translocation in the Endoplasmic Reticulum 
Michael Kowarik, Stephanie Küng, Bruno Martoglio, Ari Helenius 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1
The Cp Domain of the SFV C-Protein and Some of the Constructs Generated
(A) Crystal structure of the Semliki Forest virus capsid protease (Cp) (Choi et al., 1997). It shows the active site cleft at the contact interface of the two subdomains. Catalytic residues (H145, D167, and S219) are shown as ball-and-stick representations (green); the C-terminal tryptophan (magenta) within close proximity to the active site illustrates the substrate inhibition responsible for the “one-shot” and “cis” activity of Cp proteolysis. The most N-terminal residue visible in the crystal structure is C119 (yellow).
(B) Linear sequence of fusion proteins containing Cp. Same color code as in (A) except where indicated. The black arrow illustrates how the catalytic triad (H145, D167, and S219) comes together and points toward the Cp C terminus (W267), i.e., the substrate site for autoproteolysis. Numbers correlate with the position in the full-length, wild-type SFV capsid protein. The signal sequence is shown in yellow. Amino acid sequences of the different linker peptides are indicated. The first three residues after the Cp cleavage site (SAP) from the wild-type context were the same in all fusion proteins to ensure proper cleavage activity.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 Cp Must Fully Emerge from the Ribosomal Exit Tunnel to Fold
Autoradiographs of SDS-PAGE are shown.
(A) Truncated ssCp.pL mRNA coding for the signal sequence containing Cp and C-terminal extensions from 28 to 52 amino acids were translated in wheat germ extract. Band identities are indicated. The closed and open circles show ssCp.pL28 and ssCp.pL32, respectively. Numbers above the gel frame define the length of the linker peptide in amino acids.
(B) Samples from the experiment in (A) were treated with 5 mM puromycin instead of stabilization before lysis.
(C) Quantitation of the continuous increase in the Cp band intensity with increasing linker length, presented as the cleavage ratio of cleaved/(cleaved + uncleaved) versus linker peptide length.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Signal Sequence Processing Precedes Cp Self-Cleavage during Cotranslational Translocation
ssCp.pL60 was translated in reticulocyte lysate in the absence (lane 1) and presence (lanes 3 and 5) of microsomes and treated with 5 mM puromycin to release ribosome-bound nascent chains (lane 5). All samples obtained with truncated mRNAs (lanes 1, 3, and 5) were treated to inhibit postlysis folding. Reference peptides representing the signal sequence containing (lane 2) and lacking (lane 4) Cp*.pL60 full-length protein were derived from translating mRNA with a stop codon at the 3′ end for proper chain termination. Marker protein sizes are indicated to the right of the gel frame.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
ssCp Nascent Chains Have to Emerge from the Translocon Pore to Fold
(A) Synchronized reticulocyte lysate translations programmed with ssCp.pL mRNAs were performed in the presence of microsomes as described and analyzed by SDS-PAGE and autoradiography. Linker lengths are depicted above the gel frame. Some ssCp appeared in every sample, probably due to untargeted protein unspecifically associated with the microsomes.
(B) Samples from (A) treated with 5 mM puromycin instead of Mg2+ and processed identically.
(C) Quantitation of the molar ratio of cleaved/(cleaved + uncleaved) Cp plotted against the linker length (three experiments). Data from experiments performed with pL- and polyAla-linkers from 28 to 84 and 60 to 102 amino acids, respectively, are shown (RL, reticulocyte lysate; WGE, wheat germ extract).
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 Signal-Anchored Cp Folds Partly within the Translocon Pore
(A) Proteinase K protection assays of saCp.pL50 (left panel) and saCp.pL74 (right panel). Translations were carried out in reticulocyte lysate. Treatments are indicated above the lanes. The protein species that had lost the cytosolic tail of the signal anchor is indicated as mCp.
(B) saCp.pL polypeptides were synthesized with truncations from positions 30 to 86 downstream of the Cp C terminus. Synchronized in vitro translation/translocations were carried out in reticulocyte lysate in the presence of microsomes.
(C) Quantitation of cleavage in the saCp.pL series. For comparison, cleavage ratios of the ssCp.pL series (with and without microsomes, NTRM) from Figures 2C and 4C were included in the graph.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Cp Cleavage Is Hardly Affected Upon Shortening of the Distance from the Transmembrane Sequence to the Cp N Terminus
(A) saCp.pL74 constructs without and with a shortened linker between the N-terminal signal anchor sequence and Cp. The different amino acid sequences where the signal anchor and the Cp were fused are shown below the scheme. sa-tm abbreviates the part of the signal anchor that was not shown in full sequence detail. “FL” in bold letters are the last residues of the putative transmembrane region (Lipp and Dobberstein, 1988); “RS” (underlined) were introduced due to cloning reasons; the single “M” is the first amino acid in Cp; “CIF” in bold to the right are the first amino acids visible in the Cp crystal structure (Choi et al., 1997). Two deletion mutants that positioned the Cp domain closer to the membrane are shown below.
(B) Proteinase K protection assays. Translations were carried out as in the experiment in Figure 5A.
(C) Cleavage ratios from (B) are shown. Importantly, they did not change before and after PK treatment, nor were they significantly changed by deletions in the linker to the transmembrane sequence.
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
Model of the Ribosome/Translocon Complex Engaged in Cotranslational Translocation and Folding of Cp
The shapes of the ribosome (gray) and the translocon (magenta) have been adapted from the cryo-EM structure by Menetret et al. (2000). It indicates that the length of the ribosomal exit tunnel is 100 Å (Morgan et al., 2000), that there is a gap between ribosome and translocon of about 20 Å, that the translocon is 45 Å thick, and that there is a finger-like protrusion 50 Å away from the membrane, possibly containing elements from OST or TRAM (Menetret et al., 2000). The lipid bilayer is represented in light blue. The nascent Cp with a linker of 68 residues (dark blue), from which the signal peptide has been removed, is shown within the ribosomal exit tunnel and the translocon pore. The Cp domain is illustrated in its folded native conformation but still connected to the peptidyltransferase center (Choi et al., 1997), i.e., immediately before cleavage. In the absence of microsomes, we found that Cp folded with a linker of 39–40 amino acids, consistent with in vivo data on Cp folding (Nicola et al., 1999) and with protease protection studies (Matlack and Walter, 1995). Results from this study (italic), moreover, showed that after signal sequence cleavage, Cp folding occurred with a linker of 67–68 amino acids. About 70 residues are protease protected by the ribosome/translocon complex (Matlack and Walter, 1995), glycosylation is possible 65 amino acids from the peptidyltransferase center (Whitley et al., 1996), and 70 is the last position distal to the peptidyltransferase center that can be cross-linked to Sec61α (Mothes et al., 1994). Thus, when passing the OST and the mouth of the translocation pore, the Cp domain enters a space wide enough to allow folding. Our data suggest that the polypeptide can emerge sideways from the translocon and that the Cp domain can fold close to the membrane.
Molecular Cell  , DOI: ( /S (02) )