Topological and mutational analysis of Saccharomyces cerevisiae Ste14p, founding member of the isoprenylcysteine carboxyl methyltransferase family

Abstract

Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein. Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology. Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed. In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions. Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin. According to this model most of the Ste14p hydrophilic regions are located in the cytosol. We have also generated ste14 mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity. Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family. Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p. This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function. Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.

Figure 1

Predicted membrane spans of Ste14p. (A) Hydropathy plot of Ste14p was generated according to the algorithm of Kyte and Doolittle (1982) with a window of 11 amino acids. Hydrophobic regions are shown in black. (B) Three topology models for Ste14p are shown, with circles indicating individual amino acids. The N and C termini of Ste14p are marked. The black circles indicate the amino acid immediately preceding the insertion site of the myc epitope tags in Ste14p-myc Q3 and I239. The asterisks mark the locations of amino acids 50, 100, 150, and 200 for orientation.

Figure 2

Ste14p-myc Q3 and I239 are functional and ER-localized. (A) Mating phenotype of Ste14p-myc Q3 and I239. Patches of the indicated MATa strains were replica-plated onto a lawn of the MATα mating tester strain SM1068 on an SD plate containing 0.3 ml of YPD. Plates were incubated at 30°C for 2 d. Strains tested were SM2926 (Δste14-3) (Δ), SM3185 (CEN STE14) (WT), SM3874 (CEN STE14::myc Q3) (Q3), and SM3876 (CEN STE14::myc I239) (I239). (B) Indirect immunofluorescence of strains expressing Ste14p-myc Q3 (top) and I239 (bottom). A single cell is shown for each construct. Fixed and permeabilized cells were stained with a 1:1000 dilution of anti-myc antiserum followed by secondary decoration with Cy3-conjugated goat anti-mouse antiserum (A and D), a 1:5000 dilution of anti-Kar2p antiserum followed by secondary decoration with FITC-conjugated goat anti-rabbit antiserum (B and E), and with DAPI (4′,6-diamino-2-phenylindole) (C and F). Strains visualized were SM3798 (2μ STE14::myc Q3) and SM3800 (2μ STE14::myc I239).

Figure 3

Protease protection of Ste14p-myc Q3 and I239. Yeast membranes were treated with proteinase K in the presence and absence of 0.4% Triton X-100. After 5 min on ice, the reactions were terminated with 1 mM PMSF and 10% trichloroacetic acid. Samples were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose. Ste14p-myc and Kar2p were detected with anti-myc and anti-Kar2p antisera, respectively. (A) SM3874 (CEN STE14::myc Q3). (B) SM3876 (CEN STE14::myc I239).

Figure 4

Diagram and detection of Ste14p-Suc2p fusions. (A) Topology models of Ste14p (4 span and 6 span), with the sites of Suc2p fusions denoted by a black circle. (B) Immunoblot of Ste14p-Suc2p fusions. Crude yeast cell extracts were incubated at 37°C in the presence or absence of 1000 U of endoglycosidase H for 12 h. The cell extracts (0.4 OD₆₀₀ cell equivalents) were resolved by 8% SDS-PAGE and transferred to nitrocellulose. Ste14p-Suc2p fusions were detected with anti-Suc2p antibodies. Lanes 1 and 2, SM2705 (Δsuc2); lanes 3 and 4, SM2894 (2 μ P_PGKSUC2); lanes 5 and 6, SM4306 [2 μ P_PGKSTE14(1-25)::SUC2]; lanes 7 and 8, SM4307 [2 μ P_PGKSTE14(1-57)::SUC2]; lanes 9 and 10, SM4308 [2 μ P_PGKSTE14(1-87)::SUC2]; lanes 11 and 12, SM4361 [2 μ P_PGKSTE14(1-113)::SUC2]; lanes 13 and 14, SM4363 [2 μ P_PGKSTE14(1-156)::SUC2]; and lanes 15 and 16, SM4309 [2 μ P_PGKSTE14(1-239)::SUC2]. (C) Introduction of the mutations N191L, P192L into Ste14p-Suc2p (239) flips the orientation of Suc2p from cytosolic to partially lumenal. Cell extracts were prepared, treated, and analyzed as in B. Lanes 1 and 2, SM2705 (Δsuc2); lanes 3 and 4, SM4309 [2 μ P_PGKSTE14(1-239)::SUC2]; and lanes 5 and 6, SM4470 [2 μ P_PGKSTE14(1-239)::SUC2, N191L, P192L].

Figure 5

Protease Protection of Ste14p-HA. (A) Topology model of Ste14p (6 span), with the site of the HA insertions denoted by a black circle. (B–E) Yeast membranes were treated with proteinase K in the presence and absence of 0.4% Triton X-100. After 5 min on ice, the reactions were terminated with 1 mM PMSF and 10% trichloroacetic acid. Samples were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose. Ste14p-HA and Kar2p were detected with anti-HA and anti-Kar2p antisera, respectively. (B) SM3191 (CEN STE14::HA V75) (C) SM3428 (CEN STE14::HA K86) (D) SM4316 (2 μ STE14::HA K110) (E) SM3429 (CEN STE14::HA G144).

Figure 6

Comparison of the methyltransferase activity, mating efficiency, and a-factor halo production in strains expressing wild-type and mutant forms of Ste14p. The data for the methyltransferase activity (A) and mating efficiency (B) of strains bearing wild-type and mutant Ste14p plasmids are shown graphically and is derived from Table 2. The a-factor halo assay (C) was performed as described in MATERIALS AND METHODS. The strains used for these assays are listed in Table 2.

Figure 7

Alignment of the ICMT family depicting the location of the ste14 mutants identified by random and site-directed mutagenesis. Alignments of S. cerevisiae Ste14p, S. pombe mam4p, X. laevis Xmam4p, 2 C. elegans open reading frames (accession numbers U88175 and U80450), a rat open reading frame (accession number AF0755595.1), and human pcCMTp are shown. Protein sequences were aligned with the use of Clustal W software (Thompson et al., 1994). The default parameters of the program were used (gap extension penalty of 0.05, gap opening penalty of 10.0, protein weight matrix - Blosum series). Black boxes denote amino acid identity and gray boxes denote amino acid similarity as determined with the Boxshade server. The bars above the sequence denote the putative membrane spans of Ste14p. The black letters above the sequence denote the random mutations described in Table 2. The site-directed mutations described in Table 2 are depicted in gray. The asterisks indicate the positions of residues N191 and P192.

Figure 8

The C-terminal region of Ste14p and its ICMT homologues is similar to several bacterial open reading frames, two phosphatidylethanolamine methyltransferases, and human and yeast reductases involved in ergosterol biosynthesis. (A) Alignment of the C-terminal region of S. cerevisiae Ste14p and its homologues (S. pombe mam4p, X. laevis Xmam4p, 2 C. elegans open reading frames [accession numbers U88175 and U80450], a rat open reading frame [accession number AF0755595.1], and human pcCMTp), open reading frames from Pseudomonas denitrificans (M62866), Bacillus subtilis (L77246), Mycobacterium tuberculosis (Z81451), Ralstonia eutropha (X98451), Synechocystis sp. (D90917), Archaeoglobus fulgidus (AAB91272), and Pyrococcus horikoshii (BAA30325), the S. cerevisiae lipid methyltransferases Pem1p and Pem2p, the sterol biosynthesis enzymes from S. cerevisiae Erg4p and Erg24p, and the human lamin B receptor, which is an Erg24p homologue. Protein sequences were aligned with the use of Clustal W software (Thompson et al., 1994). Black boxes denote amino acid identity and gray boxes denote amino acid similarity as determined with the use of Boxshade. The bar above the sequence denotes the putative membrane span of Ste14p. (B) Consensus sequence is based on the alignment shown in A. Only residues that are identical in ≥11 of the proteins in the alignment were scored as consensus residues; other residues are shown as an x. The hydrophobic region was determined by a hydropathy plot.

Figure 9

Topology model of Ste14p indicating the locations of the ste14 mutants described in this study. The six-membrane-span model for Ste14p, supported by the results of this study, is shown. In this model, the N and C termini of Ste14p extend into the cytosol. The mutations described in this study are indicated. The conserved residues found in the RHPxY-hyd-EE consensus region described in Figure 8 are indicated; identical residues are shaded black and similar residues are shaded gray.