Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

Abstract

bThe fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade.

Casein Kinase 1γ Ensures Monopolar Growth Polarity under Incomplete DNA Replication Downstream of Cds1 and Calcineurin in Fission Yeast

“…Accordingly, we sought to identify the palmitoyltransferase that is responsible for the observed Cki3 localization. As recently reported (39,40), the fission yeast genome contains five open reading frames (ORFs) that encode proteins containing the DHHC (Asp-His-His-Cys) motif, which is characteristic of palmitoyltransferase (18,41) (Fig. 6B).…”

“…6C). Erf2 requires the coactivator Erf4 for its full function (39,40), and indeed, Cki3-GFP was also delocalized from the plasma membrane in the erf4⌬ mutant (D. Hirata and T. Koyano, unpublished observation). Cki3 localization was not distinctively altered under Cds1 overproduction (see Fig.…”

“…Humans have 23 or 24 DHHC-containing proteins (42,43), and global proteomic analyses suggest that a plethora of proteins are palmitoylated in vivo (41,44,45). In budding and fission yeasts, the Erf2-Erf4 complex catalyzes modification of Ras and Rho family proteins (39,40,43,44,46,47). Interestingly, however, in budding yeast, it is the Akr1 palmitoyltransferase, not Erf2-Erf4, that is responsible for Yck2 palmitoylation (17,44).…”

Traffic Through the Trans-Golgi Network and the Endosomal System Requires Collaboration Between Exomer and Clathrin Adaptors in Fission Yeast

“…To detect membrane proteins, the cleared lysates were incubated in the presence of 1.6 M urea in a tube rotator for 16 hr at 4°and the proteins were denatured at 65°for 5 min. Primary antibodies were anti-GFP (JL8, 1:3000; BD Living Colors), anti-HA (12CA5, 1:5000; Roche), anti-a tubulin (clone B-5-1-2, 1:10,000; Sigma [Sigma Chemical], St. Louis, MO), anti-Cpy1 (clone 10A5B5, 1:100; Invitrogen, Carlsbad, CA), anti-Pep12 (clone 2C4G4, 1:500; Life Technologies), polyclonal anti-DsRed (1:1000; Clontech, #632496), polyclonal anti-Pma1 (Reyes et al 2007, 1:10,000), and polyclonal anti-human ATP6V1B2 (1:500; ABNOVA #H00000526-D01P, which recognizes fission yeast Vma2; Sanchez-Mir et al 2014). Horseradish peroxidaseconjugated anti-mouse (1:10,000; BIORAD #170-6515) and anti-rabbit (1:10,000; clone RG-96, Sigma [Sigma Chemical]) secondary antibodies were used.…”

All about that fat: Lipid modification of proteins in Cryptococcus neoformans

“…Another notable palmitoylation substrate is Rho11, the only one of the three cryptococcal Rho1 paralogs with a Cys adjacent to its CAAX box. Palmitoylation at this site likely works in conjunction with isoprenylation to localize this protein to the plasma membrane; Rho homologs in yeast and mammals that are not palmitoylated instead have a stretch of polybasic residues to serve this function (Moissoglu and Schwartz, 2014; Sanchez-Mir et al , 2014). Finally, Ras1 (the only palmitoylated protein in C. neoformans known prior to the proteomic study), was found in proteomic data sets from both wild type and the pfa4 mutant, with the expected reduction in palmitoylation in the latter.…”