title: Protein folding and maturation in a cell-free system.
abstract: Reduced cellular systems have provided important tools to study complex cellular processes. Here we describe the oxidation, oligomerization, and chaperone binding of the viral glycoprotein influenza hemagglutinin in a cell-free system. The cell-free system, comprised of rough endoplasmic reticulum derived microsomes and a reticulocyte lysate, supported the complete maturation of hemagglutinin from the earliest oxidative intermediate to the mature homo-oligomer. Hemagglutinin disulfide bond formation and oligomerization were found to occur in a time- and temperature-dependent manner. Hemagglutinin's temporal association with the molecular chaperones calnexin and calreticulin was similar to that observed for their association with elongating ribosome-attached nascent chains in live cells. Furthermore, a procedure is described that permits the translocation of protein into microsomes that are depleted of lumenal contents. This cell-free system, therefore, provided an effective means to study the biological maturation processes of a protein that traverses the secretory pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003, USA. dhebert@biochem.umass.edu
authors.forename: D N
authors.initials: DN
authors.lastname: Hebert
authors.forename: J X
authors.initials: JX
authors.lastname: Zhang
authors.forename: A
authors.initials: A
authors.lastname: Helenius
mesh_terms: D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry; D037281:Calnexin; D037282:Calreticulin; D002474:Cell-Free System; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006388:Hemagglutinins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones / Q000737:chemistry; D017510:Protein Folding; D012261:Ribonucleoproteins / Q000737:chemistry; D013696:Temperature
chemical_list: D002135:Calcium-Binding Proteins; D037282:Calreticulin; D006388:Hemagglutinins; D018832:Molecular Chaperones; D012261:Ribonucleoproteins; D037281:Calnexin