Table III.
Processing and Immunolocalization of Membrane Proteins
| Half-time of processing‡ | Localization by IIF§ | |||||
|---|---|---|---|---|---|---|
| WT | vps27Δ | |||||
| min | ||||||
| A-ALP | >180 | 70 | Golgi | |||
| (F/A)-ALP | 60 | 60 | Vacuolar | |||
| RS-ALP | 90 | 15 | Golgi | |||
| (Δ2-51)A-ALP | 85 | 15 | Golgi | |||
| (Δ2-41)A-ALP | 95 | 20 | Golgi | |||
| (Δ2-31)A-ALP | 100 | 25 | Golgi | |||
| (Δ2-21)A-ALP | 85 | 15 | Golgi | |||
| (Δ2-11)A-ALP | 90 | 15 | Golgi | |||
| (Δ68-106)A-ALP | 65 | 60 | Vacuolar | |||
| Vps10p | >180 | 20 | Golgi | |||
| Vps10p-10* | 20 | 20 | Vacuolar | |||
The kinetics of processing of the proteins listed in Table III were determined in SNY36-9A (wild-type) and NBY60 (vps27Δ) cells harboring the appropriate plasmid or producing Vps10p-10*. In brief, cells were labeled with [35S]methionine for 10 min and chased for various times by adding methionine and cysteine each to a final concentration of 50 μg/ml. Cells were spheroplasted and extracts were immunoprecipitated with a polyclonal antibody against ALP and/or Vps10p, followed by SDS-PAGE and fluorography. Half-times of processing were determined using linear regression analysis, as described in Materials and Methods.
The localization of each of the proteins was also determined in SNY36-9A (wildtype) cells by indirect immunofluorescence (IIF) using affiniity-purified polyclonal antibodies specific for ALP.