KCTD15, A NOVEL PROTEIN INVOLVED IN CELL TRAFFICKING

COLOMBO, MI; M MILAGROS LOPEZ DE ARMENTIA; ZARELLI, VEP

Congreso; Congreso Conjunto SAIB-SAMIGE 2020. Plataforma virtual.; 2020

Kctd15 belongs to a family of proteins, the Potassium Channel Tetramerization Domain (KCTD) proteins because it contains a BTB domain at the N terminal that acts as a scaffold interface. Kctd15 has been demonstrated to antagonize neural crest formation by affecting Wnt signaling and AP-2 transcription factor function during embryonic development in zebrafish. However, the function of Kctd proteins is still under characterization. Preliminary results from our lab indicates that overexpression of Kctd15 induced the formation of giant vacuoles in mammalian cells. These vacuoles do not acquire Rab5, Rab7, or CD63 or dextran internalized by endocytosis, suggesting that these compartments do not belong to the early endocytic pathway. Furthermore, they are not acidic/lysosomal structures since the vacuoles were not marked by DQ-BSA or LysoTraker. We also analyzed trans-Golgi (TGN) markers such as GFP-Rab29, GFPRab32, GFP-Rab34 and GFP-Rab38. We observed that these proteins were recruited to the membrane of the vacuoles, colocalizing totally or partially with KCTD15.We also studied the production of vacuoles by overexpressing mutated versions of KCTD15 for the SUMOylation domain. We used the K278R mutant where a lysine was mutated to an arginine at position 278, and a truncated version of KCTD15 (K234), which lacks 49 amino acids at the C-terminal, totally losing the target domain for SUMOylation. Interestingly, we observed that the mutant K278R produced vacuoles as large, or even larger, than its WT counterpart, whereas the truncated form K234 did not generate these vacuoles. This would indicate that the domain necessary for the formation of these structures seems to reside in the C-terminal region within those 49 amino acids. In order to further characterize the nature of the generated vacuoles, we used the specific marker, TGN38, one of the few known resident integral membrane proteins of the TGN. Since TGN38 moves through both the endocytic and exocytic pathways, it is useful for the identification of post-Golgi trafficking motifs. Thus, we overexpressed KCTD15 WT or the truncated form K234, tagged with FOS, observing that TGN38 colocalized with KCTD15 WT at the vacuoles membrane but not with K234 where the Golgi signal for TGN38 was still observed, indicating that the vacuoles of KCTD15 could be generated from the most posterior region of the trans Golgi. By mass spectrometry, Kctd15 was able to pull down Vps26 and Vps35, components of the retromer complex which participates in recycling components from endosomes to the trans-Golgi network (TGN). Our results show that Vps26 and KCTD15 colocalize at the vacuole while Vps35 and Vps29 (another retromer component) seem to be inside. Taken together our results suggest that Kctd15 is likely involved in the retromer-TGN trafficking and that the c-terminal domain of the protein seems to be critical for this process. Further studies are necessary to determine the role of kctd15 in this transport.