Studies on protein interaction networks in yeast have revealed that most biological processes require protein complexes rather than single proteins, and by extrapolation this also applies for mammalian cells. Interaction proteomics is thus an important approach to elucidate the functional role of the proteomes. Mass spectrometry of isolated protein complexes, protein micro-arrays, and library-based screenings, such as the yeast two-hybrid system, are the most popular current methods in interaction proteomics. While highly successful in the analysis of cytosolic and nuclear protein interactions, these approaches are of limited value to detect protein interactions on the luminal side of the organelles of the secretory pathway, since such interactions tend to be of low affinities and occur in an oxidative and ionic environment that is considerably different from that of cytosol and nucleus. To circumvent these problems we have developed a luminal yellow fluorescent protein (YFP) fragment complementation assay (PCA) to capture protein interactions inside the secretory pathway. The basic concept of PCA relies on engineering reporter protein fragments that exhibit no functional activity by themselves and do not spontaneously fold. The fragments are fused to two interacting proteins. The interaction of the hybrid proteins brings the two reporter fragments into proximity leading to their folding into the active 3D structure of the complete reporter protein. The proof of concept of the luminal YFP PCA was tested by fusing YFP fragments to the homoligomeric cargo receptor lectin ERGIC-53, its interaction partner MCFD2 and to ERGIC-53’s interacting 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. Using the YFP PCA we discovered a carbohydrate-dependent interaction between ERGIC-53 and the cathepsin Z-related protein cathepsin C. The high specificity of the assay was demonstrated by the finding that inactivation of ERGIC-53’s lectin activity by a point mutation selectively impaired YFP complementation of the carbohydrate-dependent interaction with cathepsin Z and cathepsin C. These results suggest that YFP PCA can detect weak and transient interactions inside the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion, including carbohydrate-mediated protein-protein interactions of low affinity. To search for additional proteins interacting with ERGIC-53 a genome-wide YFP PCA screening procedure was developed. A cDNA-YFP1 fusion library was constructed, expressed in COS cells and screened using YFP2-ERGIC-53 as bait. Cells expressing complemented YFP were isolated by FACS and library plasmids were recovered and analyzed. This new YFP PCA approach for the genome-wide capturing of protein interactions in living cells will be presented.