The Yeast RER2 Gene, Identified by Endoplasmic Reticulum Protein Localization Mutations, Encodescis-Prenyltransferase, a Key Enzyme in Dolichol Synthesis

(A) Underglycosylation phenotype of rer2mutants. Wild-type (WT) RER2 (SNY9) and rer2-2(SNH23-7D) cells were grown in YPD medium to 10⁷ cells/ml at 23°C and then shifted to 37°C for the indicated times. Total cell extracts were prepared, and proteins (70 μg) were analyzed by immunoblotting with the anti-Sec12p antibody. (B) Biosynthesis of CPY in rer2 mutants. The rer2-2 mutant (SNH23-7A) and wild-type (SNY9) cells were preincubated for 1 h at 23 or 37°C and then labeled for 4 min with Tran³⁵S-label and chased for the indicated times at the same temperature. CPY was immunoprecipitated from cell extracts and treated with or without endo H. Samples were analyzed by SDS-PAGE and fluorography. p1, ER form; p2, Golgi form; m, mature vacuolar form; ∗, unglycosylated pro-CPY. (C) Biosynthesis of a GPI anchor protein, Gas1p, in rer2mutants. After preincubation at 37°C for 1 h, rer2-2(SNH23-7A) and wild-type (SNY9) cells were labeled for 4 min with Tran³⁵S-label and chased at 37°C. Gas1p was immunoprecipitated and analyzed by SDS-PAGE and fluorography.

Immunofluorescence localization of an ER marker protein, BiP. Wild-type (SNY9) (A and B) and rer2-2 (SNH23-7D) (C and D) cells growing at 23°C were subjected to indirect immunofluorescence microscopy with the anti-BiP antibody. (A and C) Fluorescence images with the antibody. (B and D) DNA staining with DAPI.

Immunofluorescence localization of acis-Golgi marker protein, Ypt1p. Wild-type (SNY9) (A and B) and rer2-2 (SNH23-7D) (C and D) cells were incubated at 37°C for 4 h and subjected to indirect immunofluorescence microscopy with the anti-Ypt1p antibody. (A and C) Fluorescence images with the antibody. (B and D) DNA staining with DAPI.

Electron micrographs of rer2 cells. Wild-type (SNY9) (A) and rer2-2 (SNH23-10D) (B to E) cells were incubated at 23°C (A, B, and D) or shifted to 37°C for 2 h (C and E) and then subjected to freeze-substitution fixation and electron microscopic observation. Panels D and E are enlargements of panels B and C, respectively.

Growth phenotypes of the rer2 mutant and complementation by RER2 and SRT1. rer2-2 cells (SNH23-7D) were transformed with RER2 or SRT1 on a single-copy (CEN) or multicopy (2μm) plasmid. The transformants were streaked on MCD plates and incubated at 23 or 37°C for 4 days. The same cells were also streaked on a YPD plate containing 50 μg of hygromycin B per ml and incubated at 23°C for 4 days.

Sequence comparison of Rer2p with its homologues. The boxes show identical amino acid residues, and the borderless shading indicates similar residues. (A) Sequences of yeast Rer2p and Srt1p. (B) Rer2p homologues in various organisms, including both eukaryotes and prokaryotes. They are all hypothetical proteins found by the genome projects. The proteins are S. pombe SPAC4D7.04c, C. elegans T01G1.1, E. coli o253, H. influenzaeHI0920, B. subtilis yluA, Synechocystis strain PCC6803 SLL0506, and Methanococcus jannaschii MJ1372. Asterisks indicate the mutation points found in the rer2-1(G164D) and rer2-2 (S209N) alleles.

Disruption of the RER2 gene. (A) TheAflII-SplI region of RER2 was replaced by the LEU2 fragment. The disrupted copy of RER2was excised with HindIII and introduced into a diploid strain. (B) Tetrad analysis of the resulting strain (RER2/Δrer2::LEU2) at 23°C. All small colonies were Leu⁺, indicating that the disruption ofRER2 causes a severe growth defect.

(A) Subcellular fractionation of 3HA-Rer2p. Δrer2 cells expressing 3HA-Rer2p on a single-copy vector were spheroplasted, homogenized, and subjected to a series of centrifugations: 300 × g for 5 min, 13,000 × g for 15 min, and 100,000 × g for 45 min. Aliquots were taken from the pellet of the 13,000 × g centrifugation (P13) and the pellet (P100) and supernatant (S100) fractions of the 100,000 × g centrifugation and analyzed by immunoblotting. (B) Extraction of 3HA-Rer2p. The total homogenate was treated with the reagents indicated and centrifuged at 436,000 × g for 1 h. The pellets and supernatants were analyzed by Western blotting with the anti-HA antibody. TX-100, Triton X-100.

Subcellular localization of 3HA-Rer2p. Δrer2 cells (SMY41) harboring RER2 (A and B) or3HA-RER2 (C and D) on a single-copy plasmid were grown at 30°C and prepared for indirect immunofluorescence microscopy with the anti-HA antibody (16B12). (A and C) Fluorescence images with the antibody. (B and D) DNA staining with DAPI.

Double immunofluorescence staining of BiP and 3HA-Rer2p. Δrer2 cells (SMY41) harboring3HA-RER2 on a single-copy plasmid were grown at 30°C and prepared for indirect immunofluorescence microscopy. (A) Rhodamine fluorescence corresponding to the anti-BiP antibody. (B) Fluorescein fluorescence corresponding to the anti-HA antibody (16B12). (C) DNA staining with DAPI.

Isoprenoid biosynthesis activities in rer2mutants. Total lysates were prepared from RER2 (SNY9) (lanes 1 and 3), rer2-2 (SNH23-7D) (lanes 2 and 4), and Δrer2 (SMY20) (lane 5) cells, Δrer2 (SMY20) cells harboring RER2 on a single-copy plasmid (lane 6), andRER2 (SNY9) cells harboring RER2 on a multicopy plasmid (lane 7) and incubated with [1-¹⁴C]IPP and FPP at 20°C (lanes 1 and 2) or 30°C (lanes 3 to 7). The lipidic products were extracted with chloroform-methanol, spotted on a thin-layer chromatography plate, and developed with the benzene-ethyl acetate (95:5) solvent system (see Materials and Methods). Dolichol from porcine liver, ficaprenol (polyprenol from F. elastica), solanesol (all-trans-nonaprenpol), and squalene were used as standards.

Overexpression of RER2 suppresses the temperature-sensitive growth of sec59 cells. Thesec59 mutant (SF604-9C) was transformed with RER2on a multicopy vector, SRT1 on a multicopy vector, or a vector only. These and the control wild-type strain (ANY21) harboring an empty vector were incubated on an MCD plate at 35°C for 3 days.