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Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 450, no 4, 1587-1592 p.Article in journal (Refereed) Published
Abstract [en]

Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6(102), SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C-in, form. MRP6(102) and SP-C(Leu/Val) are inserted into the membrane in C-out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.

Place, publisher, year, edition, pages
2014. Vol. 450, no 4, 1587-1592 p.
Keyword [en]
Endoplasmic reticulum, Membrane protein topology, Protein orientation, GFP, N-linked glycosylation
National Category
Biological Sciences
URN: urn:nbn:se:su:diva-107994DOI: 10.1016/j.bbrc.2014.07.046ISI: 000341338100058OAI: diva2:752947


Available from: 2014-10-06 Created: 2014-10-06 Last updated: 2015-01-30Bibliographically approved
In thesis
1. Integration and topology of membrane proteins related to diseases
Open this publication in new window or tab >>Integration and topology of membrane proteins related to diseases
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membranes are boundaries that separate the cell from the external environment.   Membrane proteins can function as e.g. receptors and channels, allowing cells to communicate with the exterior and molecules to pass through the membrane. The biogenesis of membrane proteins involves a protein-conducting channel that aids the hydrophobic segments to partition into the membrane and translocate the hydrophilic loops. Membrane proteins need to fold to its native conformation including post-translational modifications and assembly with other proteins and/or cofactors. If this regulated pathway goes wrong the degradation machinery degrades the protein. If the system is failing can result in serious disorders. The main focus in this thesis is membrane proteins associated to diseases.

We have studied mutations in the gene of presenilin 1, which is involved in Alzheimer’s disease. We found that some mutations affect the structure and other the function of the PS1. URG7 is an unknown protein associated with liver cancer. We suggest it is localized and targeted to the ER membrane, having an NoutCin topology. SP-C is important for our lungs to function. Mutations can cause the protein to aggregate. We have studied the highly Val-rich transmembrane segment (poly-Val) and its analogue (poly-Leu) and show that poly-Leu folds into a more compact conformation than poly-Val. We show that the C-terminal chaperon-like BRICHOS domain interacts with the ER membrane, suggesting an involvement in poly-Val folding. We have also confirmed the topology of URG7, MRP6 and SP-C poly-Val/Leu using gGFP that is fused to the C-terminal of the protein.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy, 2015. 76 p.
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:su:diva-113397 (URN)978-91-7649-094-5 (ISBN)
Public defence
2015-03-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)

At the time of the doctoral defense paper 3 was unpublished and had a status as manuscript.

Available from: 2015-02-12 Created: 2015-01-29 Last updated: 2016-02-23Bibliographically approved

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