Proteomics characterization of abundant Golgi membrane proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 fr om purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold en richment over the homogenate for the Gels marker galactosyl transferase) le d to the unambiguous identification of 81 proteins including a novel Golgi- associated protein of 34 kDa (GPP34), The membrane protein complement was r esolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierar chical approach using delayed extraction matrix-assisted laser desorption i onization mass spectrometry characterization by peptide mass fingerprinting , tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Gels residents, a Gels lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-gu anine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were use d to assess the intra-Golgi and total cellular distribution of GPP34, two S NAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha (2)P24 i dentified by the proteomics approach as well as the endoplasmic reticulum c ontaminant calnexin, Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Gels complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of G PP34 predict a role for a novel coat protein in Golgi trafficking.