Involvement of GTPases and vesicle adapter proteins in Heparan sulfate biosynthesis: role of Rab1A, Rab2A and GOLPH3

Vesicle trafficking is pivotal in heparan sulfate (HS) biosynthesis, influencing its spatial and temporal regulation within distinct Golgi compartments. This regulation modulates the sulfation pattern of HS, which is crucial for governing various biological processes. Here, we investigate the effects of silencing Rab1A and Rab2A expression on the localisation of 3-O-sulfotransferase-5 (3OST5) within Golgi compartments and subsequent alterations in HS structure and levels. Interestingly, silencing Rab1A led to a shift in 3OST5 localization towards the trans-Golgi, resulting in increased HS levels within 24 and 48 h, while silencing Rab2A caused 3OST5 accumulation in the cis-Golgi, with a delayed rise in HS content observed after 48 h. Furthermore, a compensatory mechanism was evident in Rab2A-silenced cells, where increased Rab1A protein expression was detected. This suggests a dynamic interplay between Rab1A and Rab2A in maintaining the fine balance of vesicle trafficking processes involved in HS biosynthesis. Additionally, we demonstrate that the trafficking of 3OST5 in COPI vesicles is facilitated by GOLPH3 protein. These findings identify novel vesicular transport mechanisms regulating HS biosynthesis and reveal a compensatory relationship between Rab1A and Rab2A in maintaining baseline HS production.