Characterization of endoplasmic reticulum and Golgi apparatus proteins as part of cell architecture defined by in situ subcellular fractionation

• Main Authors: Yen-Chiu Chang, 張宴偢
• Other Authors: Yeou-Guang Tsay
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/29112577359562334576

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 105 === Cell biologists discovered cytoskeleton in the 20th century. By extension, the term "cytoskeleton" includes nuclear matrix, nucleoli, and extracellular matrix, forming an integrated network that connects the nucleus, cytoplasm, and extracellular matrix. Nowadays, the cytoskeleton-related research is still limited to its own anatomy, function, and structure. In Chapter II, we have collected five subcellular fractions using modified in situ subcellular fractionation. These five fractions were subjected to electrophoresis and mass spectrometry analyses. Since the remaining fraction is resistant to stringent extraction and preserves most of cell morphology, we propose to term such residual structure as cell architecture, the basic structural organization of a cell. We found that HeLa cell architecture has ~590 proteins, and among which 18% and 8% are indeed associated with the keywords endoplasmic reticulum or Golgi, respectively, based on Gene Ontology database. We have studied the protein groups removed with a four-step extraction procedure, and found that these groups have features quite distinct from the residual fraction, underscoring the unique properties of the cell architecture proteome. With information in Gene Ontology and Ingenuity Pathway Analyses databases, we identify the possible roles of this part of cell architecture in protein transport, phospholipid biosynthesis and membrane shaping. We also employed immunochemical analyses to demonstrate several ER and Golgi proteins are bona fide cell architecture proteins. For example, dolichyl-diphospho-oligosaccharide-protein glycosyltransferase 48 kDa (DDOST) subunit and the alpha-subunit of the translocon-associated protein (TRAP-α) are in the ER part of the cell architecture.