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 localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes. Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation. Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN. In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain 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 H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based 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 membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.
Watson
R T
RT
Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA.
Pessin
J E
JE
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism* / Q000648:ultrastructure; D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D015723:Gene Library; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014162:Transfection
D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins