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. 2002 May 13;157(4):631–643. doi: 10.1083/jcb.200111081

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Copyright © 2002, The Rockefeller University Press

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Figure 4. — Sec34/35 protein complex mutants are defective in Golgi glycosylation (A and B), CPY sorting (C), and recycling of resident cis-Golgi proteins (D). (A and B) Analysis of glycosylated proteins secreted by different mutants. (A) Wild-type and mutant cells were grown in YPD medium at 25°C for 18 h. The culture medium was collected and proteins concentrated by TCA precipitation. Equivalents of 100 μl of medium were loaded on 10% SDS-PAGE and Western blotted with either anti-invertase (top) or anti-Hsp150 (bottom) antibodies. (B) To analyze HSP150 secretion at the nonpermissive temperature, cells grown overnight at 25°C were washed once with water, resuspended in fresh prewarmed YPD, and incubated for 3 h at 37°C. Proteins from the culture medium were concentrated by TCA precipitation and analyzed as in A. (C) Intracellular (I) and extracellular (E) fractions were prepared from cells grown in YPD medium at 25°C for 18 h. An equivalent of 10% cell lysate and 100% of culture medium were loaded on 10% SDS-PAGE and immunoblotted with either anti-CPY or anti-Kar2 antibodies. Secretion of these proteins was quantified using the Scion Image program and the average of three independent experiments was calculated. Asterisks indicate strains that had been transformed with pYpt1. (D) v-SNAREs are not maintained in the cis-Golgi compartment in Sec34/35 complex mutants. Sec22-myc–α hybrid protein is processed by the late Golgi protease Kex2p in Sec34/35 protein complex defective strains. Immunoblot analysis of v-SNARE–myc–α proteins produced in Sec34/Sec35 protein complex mutants and RSY255 wild-type cells transformed with pWB-Acycα is shown. Aliquots of cells after an overnight incubation at 25°C in selective medium were harvested, and proteins were analyzed by immunoblotting with different antibodies as indicated. A polyclonal anti-Sec22 serum was used to detect the v-SNARE–derived hybrid proteins (uncleaved and cleaved by Kex2p protease) and the endogenous Sec22p. Monoclonal anti–c-myc antibodies were used to detect the Sec22-derived hybrid protein. Anti–α-factor antibody was used to detect only uncleaved hybrid protein.

Figure 4. — Sec34/35 protein complex mutants are defective in Golgi glycosylation (A and B), CPY sorting (C), and recycling of resident cis-Golgi proteins (D). (A and B) Analysis of glycosylated proteins secreted by different mutants. (A) Wild-type and mutant cells were grown in YPD medium at 25°C for 18 h. The culture medium was collected and proteins concentrated by TCA precipitation. Equivalents of 100 μl of medium were loaded on 10% SDS-PAGE and Western blotted with either anti-invertase (top) or anti-Hsp150 (bottom) antibodies. (B) To analyze HSP150 secretion at the nonpermissive temperature, cells grown overnight at 25°C were washed once with water, resuspended in fresh prewarmed YPD, and incubated for 3 h at 37°C. Proteins from the culture medium were concentrated by TCA precipitation and analyzed as in A. (C) Intracellular (I) and extracellular (E) fractions were prepared from cells grown in YPD medium at 25°C for 18 h. An equivalent of 10% cell lysate and 100% of culture medium were loaded on 10% SDS-PAGE and immunoblotted with either anti-CPY or anti-Kar2 antibodies. Secretion of these proteins was quantified using the Scion Image program and the average of three independent experiments was calculated. Asterisks indicate strains that had been transformed with pYpt1. (D) v-SNAREs are not maintained in the cis-Golgi compartment in Sec34/35 complex mutants. Sec22-myc–α hybrid protein is processed by the late Golgi protease Kex2p in Sec34/35 protein complex defective strains. Immunoblot analysis of v-SNARE–myc–α proteins produced in Sec34/Sec35 protein complex mutants and RSY255 wild-type cells transformed with pWB-Acycα is shown. Aliquots of cells after an overnight incubation at 25°C in selective medium were harvested, and proteins were analyzed by immunoblotting with different antibodies as indicated. A polyclonal anti-Sec22 serum was used to detect the v-SNARE–derived hybrid proteins (uncleaved and cleaved by Kex2p protease) and the endogenous Sec22p. Monoclonal anti–c-myc antibodies were used to detect the Sec22-derived hybrid protein. Anti–α-factor antibody was used to detect only uncleaved hybrid protein.