Capturing Protein Interactions in the Secretory Pathway of Living Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 102; no. 18; pp. 6350 - 6355
• Main Authors: Nyfeler, Beat, Michnick, Stephen W., Hauri, Hans-Peter, Farquhar, Marilyn Gist
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 03.05.2005; National Acad Sciences
• Subjects: Bacterial Proteins; Biological Sciences; Carrier Proteins - metabolism; Cathepsin K; Cathepsin Z; Cathepsins - metabolism; Cellular biology; Complementation; Drug interactions; Fluorescence; Fluorescent Antibody Technique; Fluorometry; Freight; Genetic Complementation Test - methods; Genetic Vectors; HeLa Cells; Humans; Immunoblotting; Lectins; Luminescent Proteins; Mannose-Binding Lectins - metabolism; Membrane Proteins - metabolism; Mutation; Principal components analysis; Protein Engineering; Protein Transport - physiology; Proteins; Proteins - metabolism; Proteins - secretion; Secretion; Secretory pathway; Transfection; Vesicular Transport Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0501976102

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 multicoagulation 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.