Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis.
We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts. Two-dimensional patterns of ghost proteins were not heavily contaminated (less than or equal to 25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8--10 fold over whole cells. Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.
D002462:Cell Membrane / Q000032:analysis* / Q000187:drug effects / Q000648:ultrastructure; D003208:Concanavalin A / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007467:Iodoproteins / Q000032:analysis; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000032:analysis*; D011402:Pronase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000032:analysis*
D007467:Iodoproteins; D008565:Membrane Proteins; D003208:Concanavalin A; D011402:Pronase