Identification of protein interaction candidates for the GOLD domain of FYCO1

Abstract

Autophagy is an evolutionary conserved degradative pathway, where damaged or surplus cytosolic components are sequestered into double membrane vesicles, autophagosomes, which become degraded through the lysosomal system. The autophagy is a dynamic process, which is depended of transport of autophagosomes along microtubule, to become degraded by lysosomes. One of the proteins involved in this transport process is FYVE and Coiled-coil [CC] domain containing protein 1 (FYCO1). FYCO1 is involved in transporting autophagosomes and late endosomes along microtubules, in the plus-end direction, by interacting with kinesin. FYCO1 interacts with membranes through phosphatidylinositol-3 phosphate via its FYVE domain. It is regulated by RAB7 interaction, via its coiled-coil region, and involved in autophagy through its interaction with LC3, via its LIR-region. No interaction partners or roles for the N-terminal RUN domain or the C-terminal GOLD domain have been revealed. Interestingly, patients with autosomal-recessive congenital cataracts have been identified with a mutation, L1376P, in the GOLD domain of FYCO1. This mutation has been suggested to link FYCO1 and human lens development and transparency together. The major aim for this study was to identify putative interaction candidates for the GOLD domain and examine the effect L1376P mutation had on the GOLD domain. From our mass spectrometry study, the GOLD domain may seem to be involved in protein-protein interactions. 182 proteins co-precipitated together with the insolated GOLD domain, but it is unknown if these interact with the GOLD domain directly or indirectly. Of these proteins, TUBA4A, DNAJA1, TXNDC5, NIPSNAP1, NIPSNAP2 (GBAS), ARF4, VPS4A, RUVBL2 and MON1B were selected for further examination. The GOLD domain showed different distribution when co-expressed with TUBA4A and VPS4A. TUBA4A was showed to be located at the centrosome in association with the GOLD domain. TUBA4A redistribute the GOLD domain to centrosomes. In addition, VPS4A was observed to localize as aggregates, and it was shown in this study that the GOLD domain may be redistributed to these VPS4A structures. It is still unclear if these interactions with the GOLD domain are indirect or direct. In addition, we studied the L1376P mutation of the GOLD domain. This mutation dramatically changes the subcellular distribution of an over-expressed GFP-GOLD domain construct from diffuse to many small aggregate-like structures. If this mutation has a similar effect on the full-length FYCO1 this may perhaps affect the transparency of the lens of carriers of this mutation.