Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-Golgi network of 3T3L1 adipocytes

To identify the targeting domains of syntaxin 6 responsible for its localiz ation to the trans-Golgi network (TGN), we examined the subcellular distrib ution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 a dipocytes, Expression of EGFP-syntaxin 6 resulted in a perinuclear distribu tion identical to endogenous syntaxin 6 as determined both by confocal fluo rescence microscopy and subcellular fractionation, Furthermore, both the en dogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment character istic of the TGN, In contrast, EGFP-syntaxin 6 constructs lacking the H2 do main were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H 2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 w as also localized to the TGN, demonstrating that the H2 domain was sufficie nt to confer TGN localization. In addition to the H2 domain, a tyrosine-bas ed plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6, Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membr ane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.