title: Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae.
abstract: In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.
authors.affiliation: Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Andy
authors.initials: A
authors.lastname: Rakestraw
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D007611:Aprotinin / Q000235:genetics; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D015815:Cell Adhesion Molecules; D002455:Cell Division; D003600:Cytosol / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D019704:Protein Disulfide-Isomerases / Q000235:genetics; D017510:Protein Folding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014617:Vacuoles / Q000378:metabolism
chemical_list: C543385:AGA2 protein, S cerevisiae; D050976:Basic-Leucine Zipper Transcription Factors; D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; C091341:HAC1 protein, S cerevisiae; D018840:HSP70 Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C060153:KAR2 protein, yeast; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C069939:a-agglutinin protein, S cerevisiae; D049452:Green Fluorescent Proteins; D007611:Aprotinin; D019704:Protein Disulfide-Isomerases