Akr1p is a PTase. (A) Purified Akr1p. Tri-tagged Akr1p was purified from detergent-treated yeast extracts with a sequence of three affinity steps. Purified protein, corresponding to an initial 2 × 10⁹ cells, was subjected to SDS-PAGE and silver staining. As a control, extracts from isogenic cells expressing the untagged, wild-type Akr1p were mock purified and stained in parallel. (B) In vitro palmitoylation. Reactions contain [³H]palmitoyl-CoA and, as indicated in the figure, 1 mM ATP, Yck2 substrate proteins purified from E. coli, and the tagged Akr1p purified from yeast. After a 60-min 30°C incubation, reactions were subjected to SDS-PAGE, fluorography, and autoradiography to assess protein labeling. The two labeled protein species were identified to be Akr1p and Yck2p. (C) Akr1p is palmitoylated in vivo. Wild-type cells transformed by either the GAL1-driven 6xHis/FLAG/HA-tagged Yck2p construct (Fig. 1) or by an analogous GAL1–AKR1 construct with a COOH-terminal HA/FLAG/6xHis tag sequence were labeled with [³H]palmitic acid and subjected to anti-FLAG IP and then SDS-PAGE, as for Fig. 1 B.

Indirect immunofluorescent localization of Akr1p. Akr1p COOH-terminally tagged with a 3xHA sequence and under control of native AKR1 upstream regulatory sequences was introduced into wild-type AKR1⁺ yeast cells on a single-copy vector plasmid (pRS316 based). Three deconvolved optical sections of the same cell are shown together with the cell visualized by DIC.

Akr1p is required for Yck2p palmitoylation. Wild-type Yck2p or one of three Yck2p mutants, having the COOH-terminal Cys-Cys (CC) replaced by SS, CCIIS, or SCIIS, all NH₂-terminally tagged with a 6xHis/FLAG/HA sequence and under the inducible control of the GAL1 promoter were introduced into wild-type (AKR1⁺) yeast cells or isogenic akr1Δ cells on single-copy plasmids (pRS316 based). (A) Subcellular localization of wild-type and mutant Yck2 proteins in AKR1⁺ and akr1Δ cells. Cells were subjected to a 2-h period of galactose (2%)-induced expression, followed by a 20-min period of glucose (3%)-mediated repression, time in which the newly synthesized kinases can achieve their final subcellular destinations. Detection of the Yck2 kinases used an anti-HA mAb as primary antibody, and then a Cy3-conjugated donkey anti–mouse secondary antibody. (B) [³H]palmitate labeling of wild-type and mutant Yck2 proteins in AKR1⁺ and akr1Δ cells. Cells were cultured and labeled with [³H]palmitic acid as described in the Materials and methods. Labeled Yck2p recovered by anti-FLAG IP was subjected to SDS-PAGE, fluorography, and autoradiography (top). To assess Yck2p recovery, a second portion of the anti-FLAG IP sample was subjected to anti-HA Western analysis (bottom). The differing gel mobilities are a consequence of differential phosphorylation; phosphatase treatment of wild-type Yck2p–containing extracts from AKR1⁺ cells shifts Yck2p gel mobility to a position coincident either with Yck2(SS)p or with wild-type Yck2p from akr1Δ cells (unpublished data). (C) Release of palmitate label from Yck2p by β-ME. Yck2p, labeled in vivo by [³H]palmitic acid and purified by anti-FLAG IP, was incubated for 10 min at 100°C in 2% SDS, 10% glycerol, 62.5 mM Tris, pH 6.8, containing the indicated concentrations of β-ME.

Akr1(D543A,H544A)p and Akr1(C546A)p (DH→AA and C→A, respectively) are unable to promote palmitoylation. (A) Mutant akr1 alleles fail to support the in vivo palmitoylation of Yck2p. Strains with the akr1 missense alleles replacing chromosomal AKR1, in addition to an isogenic akr1Δ and wild-type AKR1⁺ strain, were transformed by the GAL1–6xHis/FLAG/HA–YCK2 plasmid construct (Fig. 1 B). Cells were cultured, labeled with [³H]palmitic acid, and processed for anti-FLAG IP (top). Yck2p recovery from the anti-FLAG IP was assessed by anti-HA Western blotting (bottom). (B) Mutant Akr1 proteins are not palmitoylated in vivo. AKR1⁺ yeast cells transformed by plasmid constructs having either a GAL1-driven, HA/FLAG/6xHis-tagged, wild-type AKR1 (Fig. 2 C) or the equivalent DH→AA or C→A mutant versions were cultured, labeled with [³H]palmitic acid, and then subjected to anti-FLAG IP (top). Akr1p recovery after anti-FLAG IP was assessed by anti-HA Western blotting (bottom). (C) Mutant Akr1 proteins do not promote palmitoylation in vitro. Wild-type and DH→AA and C→A mutant Akr1 proteins having COOH-terminal 3xHA/6xHis tag sequences were partially purified from yeast via Ni-agarose. Recoveries from Ni-agarose of the wild-type and mutant Akr1 proteins were compared by anti-HA Western analysis (top). One portion of the labeled proteins from each in vitro palmitoylation reaction using the different Akr1 proteins was analyzed directly by SDS-PAGE to assess Akr1p autopalmitoylation (middle), and a second portion was subjected, before SDS-PAGE, first to anti-FLAG IP to isolate the FLAG-tagged Yck2 substrate protein (bottom).