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An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum
M P Schutze1, P A Peterson, M R Jackson
1Department of Immunology, Scripps Research Institute, La Jolla, CA 92037.
Abstract:
Use of alternative initiator methionines in human invariant (Ii) chain mRNA results in the synthesis of two polypeptides, Iip33 and Iip31. After synthesis both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. Subsequently, Iip31 is transported out of the ER, guiding MHC class II to the endocytic pathway, whereas Iip33, which differs by only a 16 residue extension at the N-terminus, becomes an ER resident. Mutagenesis of this extension showed that multiple arginines close to the N-terminus were responsible for ER targeting. The minimal requirements of this targeting motif were found to be two arginines (RR) located at positions 2 and 3, 3 and 4 or 4 and 5 or split by a residue at positions 2 and 4 or 3 and 5. Transplanting an RR motif onto transferrin receptor demonstrated that this motif can target other type II membrane proteins to the ER. The characteristics of this RR motif are similar to the KK ER targeting motif for type I membrane proteins. Indeed, RR-tagged transferrin receptor partially localized to the intermediate compartment, suggesting that like the KK motif, the RR motif directs the retrieval of membrane proteins to the ER via a retrograde transport pathway.
Insights
Two human invariant chain (Ii) protein isoforms, Iip33 and Iip31, are synthesized. A specific N-terminal arginine-rich motif (RR) targets Iip33 to the endoplasmic reticulum (ER), influencing protein localization.
Area of Science:
- Cell biology
- Molecular biology
- Protein trafficking
Background:
- Alternative initiator methionines in human invariant (Ii) chain mRNA produce two polypeptides: Iip33 and Iip31.
- Both isoforms are synthesized and inserted into the endoplasmic reticulum (ER) as type II membrane proteins.
Purpose of the Study:
- To investigate the ER targeting mechanism of the Iip33 isoform.
- To identify the specific sequence responsible for ER retention of Iip33.
Main Methods:
- Site-directed mutagenesis of the N-terminal extension of Iip33.
- Expression of Iip33 and modified Iip33 in cells.
- Fusion of the identified ER targeting motif to the transferrin receptor.
- Analysis of protein localization using cell imaging techniques.
Main Results:
- A 16-residue N-terminal extension in Iip33 is responsible for its ER residency.
- Mutagenesis revealed that multiple arginines near the N-terminus mediate ER targeting.
- A minimal "RR" motif (two arginines) at specific positions (2-3, 3-4, 4-5, or split) is sufficient for ER targeting.
- The RR motif can target other type II membrane proteins, like the transferrin receptor, to the ER.
- The RR motif's function is analogous to the KK motif for type I membrane proteins, suggesting retrograde transport.
Conclusions:
- The N-terminal "RR" motif is a novel ER targeting signal for type II membrane proteins.
- This motif directs protein retrieval to the ER via retrograde transport, similar to the KK motif.
- Understanding this mechanism provides insights into protein sorting and localization within the cell.