The glycosylation mapping assay
The number of residues required to span the distance between the ribosomal P-site and the active site of the oligosaccharide transferase (OST) can be conveniently measured by a previously described "glycosylation mapping" assay [10]. Ribosome-nascent chain complexes attached to the ER translocon are generated by translation, in the presence of dog pancreas microsomes, of truncated mRNA molecules coding for a membrane protein (E. coli Lep) lacking a 3' stop codon, Fig. 1A. A series of neighboring truncation points on the mRNA is chosen such that a unique Asn-Ser-Thr acceptor site for N-linked glycosylation is moved from a position ~ 60 codons to a position ~ 75 codons away from the 5' end of the mRNA, and the degree of glycosylation is measured for each truncated chain. As we have shown previously [10], glycosylation of the ribosome-attached nascent chain is generally observed when the glycosylation acceptor site is placed at a distance of ~ 65-75 residues away from the ribosomal P-site. As the maximum level of glycosylation typically observed for the truncated Lep constructs discussed below is ~ 60%, we define the "critical" number of residues (dP-O) required to span the distance between the P-site and the OST active site to be the chain length where 30% glycosylation is observed.
Ala- and Leu- but not Val- or Pro-based segments have a compact conformation both in the ribosome and the translocon
In the earlier study refereed to above [10], we found that 65 residues from the C-terminal P2 domain of wild type Lep are required to span the distance between the P-site and the OST active site, i.e. dP-O = 65. We further found that when a model hydrophobic stretch with the sequence ...QQQL17VKKKK... was inserted 15 residues downstream of the glycosylation site (i.e., in a location that is largely inside the translocon when the nascent chain is long enough to allow glycosylation, Fig. 1C), dP-O increased to 71 residues, suggesting a more compact, possibly α-helical, conformation of the L17V segment compared to the corresponding stretch from the wildtype P2 domain. In a fully extended chain (~ 3.3 Å per residue), approximately 11 residues need to be converted to an α-helix (~ 1.5 Å per residue) to account for the observed change in dP-O of ~ 6 residues.
Here, we have extended these studies, both by analyzing a wider selection of model segments (V18, A17V, P18, and the transmembrane α-helix from GpA) and by changing the position of the glycosylation site relative to the model segment such that the model segment is located within the ribosome rather than the translocon at chain lengths around the dP-O value, Fig. 1B. Results for the two Val-based constructs with the hydrophobic stretch in the ribosome (upper panel) and in the translocon (lower panel) are shown in Fig. 2A. In both cases, dP-O ≅ 64 residues, Fig. 2B, i.e., close to the value found for wild type Lep. The results for the GpA and P18 constructs are similar, Fig. 2B, with dP-O ≅ 65 residues. In contrast, the dP-O values for the L17V and A17V segments are 70-71 residues when they are present in the ribosome, and somewhat larger (~ 73 residues) for L17V in the translocon, Fig. 2B. Assuming that an increase in dP-O corresponds to a more compact conformation of the model segment, we conclude that the L17V and A17V segments have a compact, possibly helical, conformation when located in the ribosome-translocon channel, while the V18, P18, and GpA segments are more extended.
The differences in dP-O values do not correlate with the ability of the model segments to insert into the ER membrane
To assess the ability of the model segments to insert into the ER membrane (i.e., their stop-transfer function), a second glycosylation acceptor site was added at the C-terminal end of the protein (see Methods). As shown in Fig. 3A, model segments with efficient stop-transfer function will only be glycosylated on the upstream acceptor site, while those lacking stop-transfer function will be glycosylated on both sites. Since acceptor sites located close to the C-terminus of a protein are only about 30% glycosylated even if efficiently translocated into the ER lumen [11], we expect a mixture of singly and doubly glycosylated molecules in the latter case.
As seen in Fig. 3B, the L17V and V18 constructs were glycosylated on only one site, while 19% and 27% of the A17V and P18 constructs were doubly glycosylated, respectively. Protease treatment of microsomes also demonstrated that only a small fragment (corresponding to the H2-V18 region) was protected in the V18 construct while a major part (corresponding to the H2-P2 region) of wild type Lep was protected inside the microsome, Fig. 3C. Thus, although the V18 and GpA (8) segments do not appear to fold into a compact conformation inside the ribosome-translocon channel, they are efficient stop-transfer sequences. In contrast, the A17V sequence adopts a compact (possibly α-helical) conformation in the channel, but has little or no stop-transfer function. The L17V segment is both compact and an efficient stop-transfer sequence, while the P18 segment has an extended conformation (possibly forming a poly-proline II helix with a rise of 3.2 Å per residue [12], very close to the rise of ~ 3.3 Å per residue for a fully extended chain) and no stop-transfer function.
Poly-Val and poly-Leu TMH segments behave differently during integration into the ER membrane
Given that all stop-transfer sequences, once integrated into the membrane, are expected to form transmembrane α-helices [13], the extended conformation of the V18 and GpA model segments in the translocon channel was somewhat surprising. To study this further, we determined the "minimal glycosylation distance" (MGD) for the full-length V18 and A17V constructs, and also for a construct where the third Val residue from the N-terminal end of the V18 stretch had been changed to Pro.
The MGD value is defined as the minimum number of residues required to bridge the distance between the lumenal end of a hydrophobic transmembrane segment in a membrane protein and the OST active site [14], Fig. 4A. MGD measurements can be used to roughly position the lumenal end of a transmembrane segment relative to the ER membrane by comparison to MGD values for transmembrane helices where the position relative to the lipid bilayer has been derived from various biophysical experiments [15, 16]. In our previous studies, we have mainly measured MGD values for poly-Leu based sequences. As an example, the glycosylation profile for the L17V construct is shown in Fig. 4B, yielding an MGD value of 15.7 residues [14].
Since, as shown above, the A17V construct is efficiently translocated into the lumen of the microsomes, it is not expected to have a "minimal glycosylation distance". Indeed, all A17V-based glycosylation mutants tested are 60%-80% glycosylated, Fig. 4B. Interestingly, the V18 construct, which, like the L17V construct, forms a transmembrane segment, nevertheless has a glycosylation profile that is clearly distinct from that of the L17V construct: an initial drop from ~ 80% to ~ 40% glycosylation at roughly the same glycosylation distance as L17V (~ 15.5 residues) is followed by a plateau, and background levels of glycosylation are approached only at a glycosylation distance of ~ 10.5 residues. One possible interpretation is that there are two populations of V18 molecules at the time when the glycan moiety is added to the growing nascent chain: one that has a similar disposition relative to the OST active site as the L17V molecules (i.e., presumably α-helical and membrane-integrated with an MGD value of ~ 15.5 residues) and one with a significantly smaller MGD value (~ 10.5 residues).
To test this idea further, we also analyzed a construct where the third Val residue in the V18 segment was changed to Pro. We have previously shown that the introduction of a Pro residue in corresponding positions in a L23V transmembrane segment leads to a reduction in the MGD value of about 2.5 residues, presumably as a result of a break in the poly-Leu α-helix caused by the Pro residue [14]. Indeed, the initial drop in the glycosylation profile for the V18(P3) construct was ~ 2 residues, Fig. 4B, while the shift in the location of the second drop was only ~ 1 residue. This is consistent with the possibility that V18 molecules with MGD ~ 15.5 residues indeed have already formed a transmembrane α-helix at the time of glycosylation, whereas the remaining ones have not. More detailed kinetic studies will be needed to further substantiate this idea.