Fractionation of yeast organelles.
In summary, organelles of the secretory pathway can be effectively separated from one another using differential centrifugation followed by sucrose density gradient fractionation of wild-type or vesicle-accumulating mutant yeast cells. Up to 10-fold enrichment of the plasma membrane fraction is obtained, and resolution of the peak fractions of several organelles allows one to localize specific proteins to particular components of the pathway. Additionally, a highly purified population of constitutive secretory vesicles can be isolated from the 100,000 g membrane fraction of sec 6-4 cells on a Sephacryl S-1000 column. The success of this procedure is due to the homogeneous size of the vesicles and the high concentration of vesicles accumulated in the sec 6-4 cells. From other laboratories, methods have been described for the isolation of other organelles including the vacuole (Wiemken, 1975), plasma membrane (Tschopp and Schekman, 1983), and nuclei (Mann and Mecke, 1980), as well as an alternative procedure for the purification of secretory vesicles from yeast (Holcomb et al., 1987). For the localization of proteins to particular organelles the ability to lyse cells osmotically is an important improvement over the glass bead lysis procedure. The shear forces generated during glass bead lysis could potentially remove proteins from the surface of organelles that otherwise would be membrane-attached, causing them to appear soluble. Similarly, because the conditions required for stabilizing the association of a protein with a membrane can be quite variable depending on the lysis buffer, confirmation of localization using alternative schemes is prudent. With the advent of such techniques as confocal immunofluorescent microscopy and immunoelectron microscopy, effective methods for confirming localizations are becoming available.
Walworth
N C
NC
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Goud
B
B
Ruohola
H
H
Novick
P J
PJ
D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D005656:Fungal Proteins / Q000302:isolation & purification; D007202:Indicators and Reagents; D008854:Microscopy, Electron; D008970:Molecular Weight; D015388:Organelles / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000648:ultrastructure*; D013104:Spheroplasts / Q000648:ultrastructure; D014461:Ultracentrifugation / Q000379:methods
D005656:Fungal Proteins; D007202:Indicators and Reagents