Capturing protein interactions in the secretory pathway of living cells.
The secretory pathway is composed of membrane compartments specialized in protein folding, modification, transport, and sorting. Numerous transient protein-protein interactions guide the transport-competent proteins through the secretory pathway. Here we have adapted the yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) to detect protein-protein interactions in the secretory pathway of living cells. Fragments of YFP were fused to the homooligomeric cargo-receptor lectin endoplasmic reticulum Golgi intermediate compartment (ERGIC)-53, to the ERGIC-53-interacting multi-coagulation factor deficiency protein MCFD2, and to ERGIC-53's cargo glycoprotein cathepsin Z. YFP PCA analysis revealed the oligomerization of ERGIC-53 and its interaction with MCFD2, as well as its lectin-mediated interaction with cathepsin Z. Mutation of the lectin domain of ERGIC-53 selectively decreased YFP complementation with cathepsin Z. Using YFP PCA, we discovered a carbohydrate-mediated interaction between ERGIC-53 and cathepsin C. We conclude that YFP PCA can detect weak and transient protein interactions in the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion. The study extends the application of PCA to carbohydrate-mediated protein-protein interactions of low affinity.
Nyfeler
Beat
B
Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Michnick
Stephen W
SW
Hauri
Hans-Peter
HP
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000378:metabolism; D056657:Cathepsin K; D056663:Cathepsin Z; D002403:Cathepsins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005470:Fluorometry; D005816:Genetic Complementation Test / Q000379:methods; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins; D037241:Mannose-Binding Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D015202:Protein Engineering; D021381:Protein Transport / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D014162:Transfection; D033921:Vesicular Transport Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; C084004:LMAN1 protein, human; D008164:Luminescent Proteins; C475146:MCFD2 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D011506:Proteins; D033921:Vesicular Transport Proteins; C054562:yellow fluorescent protein, Bacteria; D002403:Cathepsins; C534182:CTSZ protein, human; D056663:Cathepsin Z; C534130:CTSK protein, human; D056657:Cathepsin K