The known functions of the Golgi complex include the sorting, packaging, post-translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two-dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP-fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33 alpha is a member of a conserved family of cytosolic Golgi-associated proteins with no known homology to any known functional domain or protein. Biochemical analyses show that GMx33 alpha differentially partitions into all phases of multiple detergent extractions, and two-dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33 alpha associated with the Golgi, several of which are phosphorylated. Evidence suggests that these post-translational modifications regulate its association with the Golgi. GMx33 alpha was not found on Golgi budded vesicles, and immuno-electron microscopy co-localizes GMx33 alpha to the trans-face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans-Golgi-associated proteins.

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were Ͼ99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that Ͼ90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200-to 400-fold with and 400-to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.

The known functions of the Golgi complex include the sorting, packaging, post-translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two-dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP-fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33a is a member of a conserved family of cytosolic Golgi-associated proteins with no known homology to any known functional domain or protein.Biochemical analyses show that GMx33a differentially partitions into all phases of multiple detergent extractions, and two-dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33a associated with the Golgi, several of which are phosphorylated. Evidence suggests that these posttranslational modifications regulate its association with the Golgi. GMx33a was not found on Golgi budded vesicles, and immuno-electron microscopy co-localizes GMx33a to the trans-face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans-Golgi-associated proteins.

The discovery of additional endogenous Golgi proteins will lead to significant new insights into Golgi function. To this end, stacked Golgi fractions (SGFs) were isolated from rat liver before (CTL SGF) and after molecules in transit through the Golgi were cleared by pre-treatment with cycloheximide (CHX SGF). Electron microscopic (EM) morphometric and biochemical analyses showed that the in vivo stacked morphology is retained, that > 90% of the elements can be positively identified as Golgi stacks and cisternae, and that transmembrane protein markers of the Golgi complex are enriched 300- to 800-fold over starting postnuclear supernatant (PNS). High-resolution two-dimensional (2-D) gel mapping has been carried out on the CTL PNS, CTL SII (an intermediate fraction), CTL SGF, CHX SGF, CHX SGF - high pH supernatant, and CHX SGF - high pH pellet. This analysis, coupled with immunoblotting and alignment of the 2-D gels with master gels, has allowed the identification of a number of known proteins and the preliminary characterization of the most abundant 173 Golgi-specific proteins. These 173 proteins have been placed into three categories: cargo, cytosolic Golgi-associated, and resident Golgi proteins. These categories are tentative and will be modified as more data are acquired from immunoblotting and protein sequencing.