Different conformations of nascent polypeptides during translocation across the ER membrane

Abstract

In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome. We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244).Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro). The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly α-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.

Background

In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome.

Results

We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244).Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro).

Conclusions

The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly α-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.

Background

].

V) may form individually stable α-helices already in the ribosomal channel.

The glycosylation mapping assay

) required to span the distance between the P-site and the OST active site to be the chain length where 30% glycosylation is observed.

]. (B) Truncated Lep with a model segment (white) placed in the ribosome. The glycosylation site is placed in position 178 in Lep. (C) Truncated Lep with a model segment (white) placed in the translocon. The glycosylation site is placed in position 200 in Lep. In both panels B and C, the model segment is introduced between positions 214 and 220 in Lep.

Ala- and Leu- but not Val- or Pro-based segments have a compact conformation both in the ribosome and the translocon

of ~ 6 residues.

, and GpA segments are more extended.

codons 214 and 220 in all constructs. Half-maximal glycosylation (30%) is indicated by the horizontal line.

values do not correlate with the ability of the model segments to insert into the ER membrane

], we expect a mixture of singly and doubly glycosylated molecules in the latter case.

construct (right panel). The detergent Triton X-100 was included to dissolve the microsomal membrane in lanes 4 and 8. Non-glycosylated and glycosylated molecules are indicated by a black dot and a white dot, respectively. Protease-protected fragments are indicated by a white square.

], 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

stretch had been changed to Pro.

].

].

V 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).

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.

Discussion

].

The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly α-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.

).

]), and would seem to allow the formation of larger folded structures; we see no evidence for this, however.

], where it was shown that more hydrophobic transmembrane segments move from the translocon channel into the lipid bilayer more easily (and thus presumably more rapidly) than less hydrophobic segments.

In summary, it appears that our approach using engineered glycosylation sites, truncated nascent chains, and different model polypeptide sequences makes it possible to characterize the conformational propensities of different polypeptide segments during translocation across and integration into the ER membrane. Our results also point to the necessity to carry out a careful analysis of the glycosylation profile when MGD values are used to infer the position of a transmembrane segment relative to the ER membrane.

Enzymes and chemicals

C]-methylated marker proteins, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and the cap analog m7G(5')ppp(5')G were from Amersham-Pharmacia (Uppsala, Sweden). The PCR purification and RNeasy RNA clean up kits were from Qiagen (Hilden, FRG). The PCR mutagenesis kit was from Stratagene (La Jolla, CA). Proteinase K was from Boehringer Mannheim GmbH (Mannheim, Germany). PMSF (phenylmethylsufonyl fluoride) was from ICN Biochemicals Inc (Aurora, Ohio). Puromycin was from Sigma (St. Louis, Missouri). Oligonucleotides were from Kebo Lab and Cybergene (Stockholm, Sweden).

DNA manipulations

] or by PCR. All mutants were confirmed by sequencing of plasmid DNA. All cloning steps were done according to standard procedures.

Construction of full-length and truncated Lep glycosylation mutants

] for enhanced translation and a XbaI site for cloning: ...ATAACCCTCTAGAGCCACCATGGCGAATATG...(XbaI site and initiator codon underlined). Mutants of Lep were cloned into pGEM1 behind the SP6 promoter as an XbaI-SmaI fragment.

transcription of truncated mRNA with or without a 3' stop codon were prepared using PCR to amplify fragments from pGEM1 plasmids containing the relevant Lep constructs. The 5' primer was the same for all PCR reactions and had the sequence 5'-TTCGTCCAACCAAACCGACTC-3'. This primer is situated 210 bases upstream of the translational start, and all amplified fragments thus contained the SP6 transcriptional promoter from pGEM1. The 3' primers were designed according to the desired C-terminal end of the truncated protein and either contained no stop codon or a TAG stop codon. All primers were designed to have approximately the same annealing temperature. PCR amplification was performed with a total of 30 cycles using an annealing temperature of 52 °C. The amplified DNA products were purified using the Qiagen PCR purification kit as described in the manufacturers protocol and verified on a 1.2 % agarose gel.

), the templates were amplified as above but with a 3' primer encoding the C-terminal sequence ...PTGLRLSNSTGIH(stop) corresponding to the C-terminal end of Lep but with the underlined residues changed to encode a glycosylation acceptor site.

.

Expression in vitro

] or by PCR amplification with the pGEM1 construct as template as described above. Amplified PCR fragments were transcribed from the SP6 promoter using the Large Scale RNA Synthesis kit with the RiboMAX SP6 RNA polymerase system. Transcriptions were carried out at 30°C for 12 hours. The mRNAs were purified using Qiagen RNeasy clean up kit and verified on a 1% agarose gel.

)

].

Acknowledgements

This work was supported by grants from the Swedish Cancer Foundation and the Swedish Natural and Technical Sciences Research Councils to GvH. Dog pancreas microsomes were a kind gift from Dr. M. Sakaguchi, Fukuoka.

Ismael Mingarro and IngMarie Nilsson contributed equally to this work.