Proteomics Characterization of Abundant Golgi Membrane Proteins

Abstract

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization 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 Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and ␣ 2 P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Bap31 Is an Itinerant Protein That Moves between the Peripheral Endoplasmic Reticulum (ER) and a Juxtanuclear Compartment Related to ER-associated Degradation

“…If this possibility is correct, Bap31 cycling between the ER and the recycling compartment is expected to occur by a novel nonvesicular mechanism that may be dependent on COPII and COPI. Our results also do not exclude the possibility that some Bap31 is transported to the ERGIC and/or the Golgi apparatus in certain cells, as reported previously (Bell et al, 2001). It is possible that some Bap31 escapes the cycling pathway and that it is incorporated into transport vesicles destined for the Golgi apparatus in some cells and/or under certain circumstances.…”

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

“…In-gel enzymatic digestion and Q-ToF mass spectrometry Protein bands were excised from a Coomassie blue-stained gel and destained prior to trypsin digestion (Bell et al 2001) Mass spectrometry analysis of the supernatants from the digests was done at the Montreal Network for Pharmaco-Proteomics and Structural Genomics, McGill University (Montreal, Canada). Mass spectrometric analyses were performed with a QTof II (Micromass, Manchester, UK).…”

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

“…Using methods, such as differential centrifugation, electrophoresis, or affinity fractionation, cell or tissue lysates are preseparated into fractions containing particular organelles [8,12,14]. After this prefractionation, the "organelle proteome" can be analyzed by use of established proteomics techniques, such as 1-and 2-DE and chromatographic methods followed by MS [8,[12][13][14][15][25][26][27][28].…”

“…After isolation of organelles or subcellular structures, and before electrophoretic separation and final identification by MS, fractionation by selective extraction with different agents and chromatographic separation with different resins can further facilitate proteome analysis [4,12,[23][24][25][26][27][28][29][30].…”