Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep 26;170(7):1091-9.
doi: 10.1083/jcb.200507048.

The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

Jessica E Smotrys  1 , Marissa J Schoenfish, Monica A Stutz, Maurine E Linder
Affiliations

The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

Jessica E Smotrys et al. J Cell Biol. .

Abstract

Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145-3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133-35140). Proteins that contain an Asp-His-His-Cys (DHHC)-cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
PFA3 and SWF1 promote vacuole fusion. Cells were grown in YPD or treated as indicated in minimal media (2 mM DTT for 2 h or 0.05% glucose for 4 h) and stained with FM 4–64 before microscopy. Bars, 2 μm.
Figure 2.
Figure 2.
Pfa3p-3xGFP localizes to the vacuole membrane. Cells expressing Pfa3p-3xGFP (YML163) were stained with FM 4–64 and visualized by microscopy. Bars, represent 2 μm.
Figure 3.
Figure 3.
An intact DHHC-CRD is required for PFA3 to function in vacuole fusion. (A) pfa3Δ cells transformed with vector (pRS313), PFA3-2xFlag (pML780), or C134S PFA3-2xFlag (pML782), were grown in selective media, treated with 2 mM DTT, and stained with FM 4–64 before microscopy. Pictures are representative of two independent transformants. Bars, 2 μm. (B) Whole cell lysates (50 μg) from pfa3Δ transformants used in A were probed with α-Flag antibody to detect expression of Pfa3p-2xFlag.
Figure 4.
Figure 4.
Pfa3p is required for Vac8p palmitoylation and membrane association in vivo. (A) WT, pfa3Δ, pfa3Δerf2Δ, and pfa3Δswf1Δ cells transformed with Vac8p-myc-GFP (pML735) were incubated with [3H]palmitate for 15 min before lysis and immunoprecipitation. Vac8p protein was quantitated from gels stained with SYPRO. Palmitoylation was detected by fluorography and quantitated by densitometry. Protein and [3H]palmitate values were normalized to wild type and expressed as ratios in the bar graph. (B) Whole cell lysates from WT or pfa3Δ cells transformed with Vac8p-myc-GFP underwent differential centrifugation to evaluate Vac8p association with internal membranes (IM, P13), plasma membrane (PM, P100), or cytosol (C, S100). Data shown in both A and B are representative of at least two experiments performed with independent transformants.
Figure 5.
Figure 5.
Pfa3p promotes Vac8p palmitoylation in membranes. Pfa3p-Flag (WT [pML395] or C134S[pML724]) and Vac8p-myc (WT [pML734] or Cys− [pML775]) constructs were coexpressed in yeast and membranes incubated with [3H]palm-CoA for 10 min and analyzed by fluorography or subjected to Western blot to evaluate protein expression. The result shown is representative of four experiments. The radiolabeled samples were exposed to film for 10 d.
Figure 6.
Figure 6.
Vac8p is selectively palmitoylated by Pfa3p. Membranes expressing Pfa3p-Flag, Yol003p-Flag, Ydr459p-Flag, Flag-Erf2p/GST-Erf4p, or Akr1p-Flag were incubated with 3.0 μg myr-Vac8p-myc-6xHis and [3H]palm-CoA for 10 min and analyzed by fluorography (5 d exposure). Data are representative of two independent experiments.
Figure 7.
Figure 7.
Partially purified Pfa3p-Flag autoacylates and palmitoylates Vac8p. Detergent extracts from Pfa3p-Flag–expressing cells or vector-transformed cells (pESC) were purified by Flag-immunoaffinity chromatography. Equal volumes of Pfa3p-Flag and pESC elutions were analyzed by (A) silver stain, (B) α-Flag Western blot, or (C) fluorography after incubation with [3H]palm-CoA. (D) Pfa3p-Flag (0.5 pmol) and myr-Vac8p-myc-6xHis (0.4 μg total protein/3 pmol Vac8p) were incubated with [3H]palm-CoA for the indicated time and analyzed by fluorography (inset; 4 d exposure) and scintillation counting (curve). Data are representative of two independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adams, A., D.E. Gottschling, C.A. Kaiser, and T. Stearns. 1997. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Plainview, NY. 177 pp.
    1. Bohm, S., D. Frishman, and H.W. Mewes. 1997. Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins. Nucleic Acids Res. 25:2464–2469. - PMC - PubMed
    1. Bucci, M., and S.R. Wente. 1997. In vivo dynamics of nuclear pore complexes in yeast. J. Cell Biol. 136:1185–1199. - PMC - PubMed
    1. Chen, D.C., B.C. Yang, and T.T. Kuo. 1992. One-step transformation of yeast in stationary phase. Curr. Genet. 21:83–84. - PubMed
    1. Dietrich, L.E., R. Gurezka, M. Veit, and C. Ungermann. 2004. The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion. EMBO J. 23:45–53. - PMC - PubMed

Publication types

MeSH terms