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Protein folding within and protein transport into mammalian microsomes are differentially affected by photoaffinity
M Brunke1, J Tyedmers, R Zimmermann
1Universität des Saarlandes, Homburg, Germany.
Abstract:
Transport of presecretory proteins into mammalian microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with 8-azido-ATP. Typically, protein folding within the lumen of the endoplasmic reticulum of mammalian cells depends on ATP and the member of the Hsp70 protein family, BiP. Here we addressed the question of whether protein transport into and folding within microsomes are differentially affected by photoaffinity labeling of microsomes with 8-azido-ATP. Folding of heterodimeric luciferase to the native state was more azido-ATP-sensitive compared to transport of the precursors of the two subunits. Therefore, we conclude that the microsomal protein which is responsible for the ATP-dependence of protein folding in the endoplasmic reticulum is sensitive to photoaffinity labeling with 8-azido-ATP and that this microsomal protein is distinct from the microsomal ATP-binding protein which is involved in protein transport.
Insights
Photoaffinity labeling with 8-azido-ATP distinguishes protein folding from transport in mammalian microsomes. A specific microsomal protein involved in ATP-dependent protein folding is distinct from the ATP-binding protein mediating transport.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein transport into mammalian microsomes is crucial for cellular function.
- Endoplasmic reticulum protein folding relies on ATP and heat shock protein 70 (Hsp70) family member, BiP.
- The role of ATP-dependent microsomal proteins in distinct processes like transport and folding requires clarification.
Purpose of the Study:
- To investigate whether protein transport into microsomes and protein folding within microsomes are differentially affected by photoaffinity labeling with 8-azido-ATP.
- To identify distinct microsomal ATP-binding proteins involved in protein transport versus protein folding.
Main Methods:
- Utilized photoaffinity labeling with 8-azido-ATP on mammalian microsomes.
- Assessed the sensitivity of presecretory protein transport and heterodimeric luciferase folding to 8-azido-ATP labeling.
- Compared the effects of labeling on protein transport and protein folding.
Main Results:
- Photoaffinity labeling with 8-azido-ATP differentially affected protein folding and transport.
- Folding of heterodimeric luciferase was more sensitive to 8-azido-ATP labeling than the transport of its precursor subunits.
- This indicates distinct ATP-dependent mechanisms for protein folding and transport.
Conclusions:
- A specific microsomal protein responsible for ATP-dependent protein folding in the endoplasmic reticulum is sensitive to 8-azido-ATP photoaffinity labeling.
- This protein is distinct from the microsomal ATP-binding protein involved in protein transport.
- The study differentiates the molecular machinery for protein folding and transport within microsomes.