Functional oligomerization of the Saccharomyces cerevisiae isoprenylcysteine carboxyl methyltransferase, Ste14p.
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-terminal-tagged 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.
Griggs
Amy M
AM
Department of Chemistry and the Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.
Hahne
Kalub
K
Hrycyna
Christine A
CA
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D003432:Cross-Linking Reagents; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D020125:Mutation, Missense; D011496:Protein Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013388:Succinimides
D003432:Cross-Linking Reagents; D029701:Saccharomyces cerevisiae Proteins; D013388:Succinimides; D011496:Protein Methyltransferases; C498863:Ste14 protein, S cerevisiae