Ubiquitination and Degradation of the Zebrafish Paired‐Like Homeobox Protein Vsx‐1

: Vsx‐1 is a paired‐like : CVC homeobox protein dynamically expressed during zebrafish development. Previous results indicate that Vsx‐1 influences bipolar cell differentiation and maintenance of these cells in the adult retina. To understand the developmental regulation of this transcription factor, we investigated ubiquitination as a possible posttranslational mechanism. In vitro, Vsx‐1 was conjugated with multiple ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the accumulation of Vsx‐1‐ubiquitinn complexes and stabilized unmodified Vsx‐1. Also, in transiently transfected COS‐7 cells, Vsx‐1 is ubiquitinated, and pulse‐chase experiments show that Vsx‐1 proteolysis occurs. Vsx‐1 proteins with C‐terminal deletions retained the capacity for initial modification by ubiquitin but lost the capacity for efficient chain elongation. These results show that Vsx‐1 is a substrate of the ubiquitin/proteasome pathway and suggest that C‐terminal sequences of Vsx‐1 are critical for ubiquitin chain elongation. In addition, our findings suggest that ubiquitin‐dependent proteolysis regulates Vsx‐1 during zebrafish retinal development.