GMx33: A novel family of trans-Golgi proteins identified by proteomics

The known functions of the Golgi complex include the sorting, packaging, po st-translational modification, and transport of secretory proteins, membran e proteins, and lipids. Other functions still remain elusive to cell biolog ists. With the goal of identifying novel Golgi proteins, a proteomics proje ct was undertaken to map the major proteins of the organelle using two-dime nsional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP-fusion constructs. Multiple unknown s were identified, and the initial characterization of one of these protein s is reported here. GMx33 alpha is a member of a conserved family of cytoso lic Golgi-associated proteins with no known homology to any known functiona l domain or protein. Biochemical analyses show that GMx33 alpha differentia lly partitions into all phases of multiple detergent extractions, and two-d imensional immunoblots reveal that there are multiple differentially modifi ed forms of GMx33 alpha associated with the Golgi, several of which are pho sphorylated. Evidence suggests that these posttranslational modifications r egulate its association with the Golgi. GMx33 alpha was not found on Golgi budded vesicles, and immune-electron microscopy co-localizes GMx33 alpha to the trans-face on the same three cisternae as TGN38 in normal rat kidney c ells. This work represents the preliminary characterization of a novel fami ly of trans-Golgi-associated proteins.