Most membrane proteins in eukaryotic cells are co-translationally
integrated into the endoplasmic reticulum (ER) membrane at aqueous pores
termed translocons. During multi-spanning membrane protein (MSMP)
integration, the nascent polypeptide is threaded into the translocon pore where
each successive transmembrane segment (TMS) is moved laterally through the
translocon into the bilayer. The hydrophilic polypeptide segments on each side
of the TMS are alternately directed into either the aqueous cytosol or the
aqueous ER lumen. How is the ER membrane permeability barrier maintained
during this process?
For a single-spanning signal-cleaved membrane protein, nascent chain
movement into the lumen occurs while an ion-tight ribosome-translocon junction
prevents ion flow through the translocon pore. Prior to opening this junction to
allow nascent chain movement into the cytosol, BiP (Hsp70 binding protein)
effects closure at the lumenal end of the pore to maintain the membrane
permeability barrier. To determine whether the ribosome and BiP alternately mediate pore closure during the integration of a MSMP, integration
intermediates with nascent chains of different lengths were prepared with a
fluorescent probe positioned in the nascent chain far inside the ribosomal tunnel.
Nascent chain exposure to the cytosol or lumen was then detected by the
collisional quenching of the probe by iodide ions located on either the cytosolic
or lumenal side of the membrane.
While the first TMS through the tunnel caused the ribosome-translocon
junction to open, the second TMS elicited both the closure of this junction and
the opening of the lumenal end of the pore. Movement of a third TMS through
the tunnel caused the ribosome-translocon junction to re-open after closure of
the lumenal end. Pore opening and closing occurred after each TMS was 4-7
residues from the peptidyltransferase center, irrespective of TMS location in the
nascent chain. The ribosome treated all TMSs in the same manner, regardless
of their individual sequence or their native orientation. The ER membrane
permeability barrier is maintained by ribosome-translocon interactions during cotranslational
MSMP integration.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2894 |
| Date | 15 May 2009 |
| Creators | Jongsma, Candice Gene |
| Contributors | JOHNSON, ARTHUR E. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
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