Functional Oligomerization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxyl Methyltransferase, Ste14p

Abstract

The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26-kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CAAX post-translational processing pathway. Sequence analysis reveals a putative GXXXG dimerization motif located in transmembrane 1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminaltagged His 10 -myc 3 -Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species.Post-translational modifications of eukaryotic proteins have profound effects on their localization and function. Many eukaryotic proteins are modified at their C termini by key modifications including the addition of an isoprenyl lipid moiety, proteolysis, and methylation (1-5). The consensus sequence for isoprenylation, proteolysis, and methylation is a C-terminal CAAX sequence where C is cysteine, A is an aliphatic residue, and X can be one of several amino acids. This three-step posttranslational processing has been recognized as crucial for the proper membrane localization and subsequent biological function of Ras proteins and many other key signal transduction proteins, including the Rho small GTP-binding proteins (2, 4, 6, 7).The final modification, ␣-carboxyl methyl-esterification of the newly exposed cysteine carboxyl group is performed by an isoprenylcysteine carboxyl methyltransferase (Icmt) 3 (4, 8 -18).Ste14p from Saccharomyces cerevisiae is the founding member of the Icmt family. A 239-amino acid enzyme with a molecular mass of 26 kDa, Ste14p has six transmembrane-spanning segments and is localized to the endoplasmic reticulum membrane (13,19). In yeast, carboxyl methylation has been shown to be important for both the proper cellular localization of RAS proteins and the formation of active a-factor mating pheromone (9, 13, 14). Ste14p possesses a tandem 31 GXXXGXXXG 39 motif located in transmembrane segment one. The GXXXG motif has been shown to be important in the dimerization of several integral single and multispanning membrane proteins (20, 21) including glycophorin A (22, 23), the ABC transporter ABCG2 (24), yeast ATP synthase (25), human carbonic anhydrase (26), Helicobacter pylori vacuolating toxin (27), and yeast ␣-factor receptor (28). The spacing of the glycine residues allows them to be positioned on the same face of the helix, and it is believed that the sma...

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

“…Residues on M1 and M3 that make contacts between these helices are drawn as sticks and coloured magenta. Because the M1 and M2 helices are not present in Saccharomyces cerevisiae ICMT enzyme, we hypothesize that the GxxxG motif that is present in the first transmembrane helix of yeast ICMT (equivalent to M3 of beetle ICMT) may cause dimerization of the yeast enzyme through packing of these helices 41 , whereas ICMT enzymes that contain M1 and M2, which includes human and beetle ICMT, are monomeric. The location of a PreScission protease cleavage site (PS site) that was inserted in the connection between the M2 and M3 helices (introduced at Asn58) for experiments outlined in this figure is indicated.…”

Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites

“…dimerization) through interactions of helical transmembrane domains (28). Although the oligomeric state of ICMT has not been definitively resolved, it has been suggested that it may function as a dimer or higher order oligomer (29). To determine the importance of the residues in the GXXXG-like motif for ICMT activity, we mutated three residues of Ag ICMT in this motif (Ala-68, Gly-72, and Ser-76).…”

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

“…Recent evidence suggests that Ste14 forms a dimer or other higher-order oligomer through a GXXXG motif in its first transmembrane span (104,218). Heterologous expression of either the Schizosaccharomyces pombe STE14 homolog, Mam4, or the mammalian ICMT can functionally complement a yeast ste14⌬ mutant, indicating that distant homologs have conserved function (68,220).…”