Traffic 2009; 10: 1061–1073© 2009 John Wiley & Sons A/S

doi: 10.1111/j.1600-0854.2009.00925.x

Analysis of DHHC Acyltransferases ImpliesOverlapping Substrate Specificity anda Two-Step Reaction Mechanism

Haitong Hou1, Arun T. John Peter1, Christoph

Meiringer1,2, Kanagaraj Subramanian1,3 and

Christian Ungermann1,*

1University of Osnabruck, Department of Biology,Biochemistry section, Barbarastrasse 13, 49076Osnabruck, Germany2Present address: Roche Diagnostics GmbH, 82377Penzberg, Germany3Present address: The Scripps Institute, 10550 NorthTorrey Pines Road, La Jolla, CA 92037, USA∗Corresponding author: Christian Ungermann,Christian.Ungermann@biologie.uni-osnabrueck.de

Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD)

acyltransferases are polytopic transmembrane proteins

that are found along the endomembrane system of

eukaryotic cells and mediate palmitoylation of peripheral

and integral membrane proteins. Here, we address the

in vivo substrate specificity of five of the seven DHHC

acyltransferases for peripheral membrane proteins by an

overexpression approach. For all analysed DHHC proteins

we detect strongly overlapping substrate specificity.

In addition, we now show acyltransferase activity for

Pfa5. More importantly, the DHHC protein Pfa3 is able

to trap several substrates at the vacuole. For Pfa3 and

its substrate Vac8, we can distinguish two consecutive

steps in the acylation reaction: an initial binding that

occurs independently of its central cysteine in the DHHC

box, but requires myristoylation of its substrate Vac8,

and a DHHC-motif dependent acylation. Our data also

suggest that proteins can be palmitoylated on several

organelles. Thus, the intracellular distribution of DHHC

proteins provides an acyltransferase network, which may

promote dynamic membrane association of substrate

proteins.

Key words: acyltransferase, DHHC, palmitoylation, Pfa3,

Vac8

Received 5 May 2008, revised and accepted for publi-

cation 15 April 2009, uncorrected manuscript published

online 21 April 2009, published online 12 May 2009

Function of proteins in the endomembrane systemis largely determined by their subcellular localization.Membrane association of peripheral membrane proteinsmay be facilitated by positively charged amino acidstretches or the covalent addition of lipids (like prenylationor myristoylation), although the binding affinity tomembranes mediated by these interactions is weak (1,2).

An alternative means of membrane association isprotein palmitoylation (3). The thioester bond betweenthe palmitate and the cysteine of the target proteinis generated with the help of acyltransferases (4,5).A family of polytopic transmembrane proteins, namedDHHC cysteine-rich domain (CRD) proteins, according toa consensus sequence in their cytosolic domain, has beenidentified to function as acyltransferases in yeast andother eukaryotes (3,6). Whereas 23 DHHC isoforms havebeen found in humans, only 7 are present in yeast. Theyare located on distinct membranes of the endomembranesystem; three proteins (Erf2, Swf1 and Pfa4) localize to theendoplasmic reticulum (ER), two on the Golgi (Akr1 andAkr2), one on the vacuole (Pfa3) and Pfa5 is present on theplasma membrane (7). On the basis of deletion analyses,substrate-specific activities have been demonstrated forErf2 towards Ras2 (8), for Swf1 towards the SNARETlg1 (9), for Akr1 towards Yck1/2 and Lcb4 (10,11), forPfa4 towards the chitin synthase Chs3 (12) and for Pfa3towards the vacuolar fusion factor Vac8 (13,14). So far,neither activity nor substrates have been described forPfa5.

Using knock-out strains of up to six DHHC proteins,Davis and co-workers conducted a proteomic surveyto identify palmitoylated proteins and their connectionto DHHC proteins (15). A large portion of palmitoylatedperipheral membrane proteins can be subdivided intothree groups according to the positions of palmitoylatedcysteines; those that have cysteines proximal to the N-terminal myristolyation motif (e.g. Vac8; I), adjacent tothe C-terminal prenylation sequence (e.g. Ras2; II) andnear the C-terminus of the protein (e.g. Yck3, Yck2; III).Most of these proteins are either found at the plasmamembrane or the vacuole. Group I proteins and alsosome members of group II are mostly dependent onErf2. Some depend on Akr1, whereas others could not beassigned to any specific DHHC. This observation suggeststhat multiple DHHCs could have overlapping specificities,although this has not been shown so far. In fact, it has notbeen possible to identify a clear consensus sequence forpalmitoylation (15).

Here, we present evidence that five of the DHHCproteins when overexpressed mediate palmitoylationand localization of several peripheral membrane proteinsubstrates such as the vacuolar fusion factor Vac8,the casein kinases Yck1-3 or Ras2. We also find thatPfa5 has acyltransferase activity. For Pfa3, we provideevidence for a two-step reaction mechanism, whichincludes substrate binding. Our data suggest that the

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endomembrane distribution of multiple DHHCs with abroad substrate specificity provides a framework for thelocalization and modification of palmitoylated proteins.

Results

Expression and localization of overexpressed DHHCs

Upon deletion of Pfa3, the vacuole localization of Vac8,a palmitoylated peripheral membrane protein, is reduced,suggesting that it acts as the main acyltransferase forVac8 (13,14). Interestingly, the mislocalization of Vac8to the cytosol is enhanced upon a combined deletionof AKR1, AKR2, PFA3, PFA4 and PFA5 (akr1� akr2�

pfa3� pfa4� pfa5�; termed 5 × � in the remainingtext; Figure 1A,B), and Vac8 is poorly acylated underthese conditions (15). This suggests that several DHHCproteins contribute to Vac8 localization, although thiswas actually not shown previously (15). We thereforedecided to test this hypothesis directly and placedselected DHHC proteins, which localize to ER, Golgi,plasma membrane and endosome/lysosome, under thecontrol of the galactose promotor and tagged them C-terminally with Protein A or green fluorescent protein(GFP) (Figure 2). Akr2 was not tested because of itslethality upon overproduction (16).

Upon induction in galactose, overexpression of all DHHCswas detected (Figure 2A). Pfa3 was then expressed at acomparable level as its substrate Vac8. Our quantificationindicates that wild-type cells contain 10-fold more Vac8than Pfa3 (Figure 2B), indicating a 10-fold overproductionof Pfa3 in our overexpression strain. Akr1 overexpressionlevels were similar to Pfa3, whereas Erf2, Pfa4 andPfa5 expression was significantly lower (Figure 2A). Weanalysed the subcellular localization of the overexpressedDHHCs by fluorescence microscopy. Despite their correctlocalization to the ER (Pfa4, Erf2), plasma membrane (Pfa5)and vacuole (Pfa3), a significant portion of GFP-taggedDHHCs was localized to the vacuole lumen, which mightbe a result of the overproduction (Figure 2C). Akr1 wasfound in multiple dots that could correspond to the Golgi.

Several DHHC proteins can palmitoylate Vac8

We then tested whether overproduction of these DHHCswould be sufficient to localize selected substrates to theirtarget organelle. Initially, we focused on the vacuolar Vac8fusion protein. Vac8 is myristoylated at the N-terminalglycine and contains three cysteines at position 4, 5 and7 that can be palmitoylated and thus support membranelocalization (17–19). Previous studies suggested thatPfa3 (13,14), and Akr1 are involved in Vac8 palmitoyla-tion (15), even though a deletion of Akr1 alone shows noeffect on Vac8 membrane localization (Figure 1A). To oursurprise, any of our overexpressed DHHC was sufficientto confer vacuole membrane localization (Figure 3A,D)and palmitoylation on Vac8 (Figure 3E). In addition, thisindicates that DHHC proteins are functional despite theiroverexpression and C-terminal tagging. Interestingly,

Figure 1: Vac8 localization in cells lacking DHHC proteins. A)Cells with the indicated DHHC deletions expressing genomicallyintegrated Vac8-GFP were analysed by fluorescence microscopy.4 × � = akr2� pfa3� pfa4� pfa5�; 5 × � = akr1� akr2� pfa3�

pfa4� pfa5�. Size bar: 10 μm. B) Subcellular fractionation.Cells containing untagged Vac8 were lysed and separated asdescribed in Methods. The total cell lysate (T) corresponds tothe same amount used for the separation of membrane pellet(P) and supernatant (S). Proteins were analysed by SDS-PAGEand western blot using antibodies against the vacuolar V-ATPasesubunit Vma6, Vac8 and Arc1, a cytosolic marker protein.

even the overexpression of Erf2, which is already presentat endogenous levels in the 5x� strain, was sufficient torescue Vac8 localization and palmitoylation. Our data alsoshow that Pfa5 has acyltransferase activity (Figures 3–5).Thus, Vac8 vacuole localization and palmitoylation appearsto occur independently of the localization of the DHHCprotein and its identity.

We then tested for the specificity of this reaction. Local-ization of Vac8 required its N-terminal cysteines. A mutantlacking the cysteines (Vac8 C4,5,7A) was primarilycytosolic (Figure 3B). The Vac8-GFP membrane staining

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Figure 2: Overexpression and localization of DHHC proteins.

A) DHHC overexpression level. Total cell lysates were preparedfrom BJ VAC8-TAP and the 5 × � strains expressing the ProteinA-tagged DHHC proteins. Proteins were analysed by SDS-PAGEand western blotting using antibodies against the Protein Atag or Vti1. B) Determination of the Vac8 and Pfa3 expressionlevel in wild-type cells. Indicated amounts of lysate of BJ3505cells expressing chromosomally TAP-tagged Vac8 and Pfa3 wereanalysed by SDS-PAGE and western blot with antibodies againstthe Protein A tag and Vti1. C) Intracellular localization of GFP-tagged DHHC proteins. Cells were grown to mid-log phase andanalysed by fluorescence microscopy as described in Methods.Size bar: 10 μm.

observed in some overexpression backgrounds corre-sponds most likely to the ER, and Vac8 is known to bindthe ER protein Nvj1 (20). Indeed, a similar localization ofVac8 is already observed in the 5 × � mutant (Figure 3A).Moreover, loss of Vac8 myristoylation (Vac8 G2A) ledto a complete mislocalization of Vac8 to the cytosol(Figure 3C). This indicates that myristoylation alone mightallow for some association of Vac8 with membranesas previously observed (17). Interestingly, only Pfa3overproduction rescued the localization of either Vac8mutant, which might be because of its direct interactionand subsequent efficient palmitoylation (see below).

Palmitoylation of multiple substrates by DHHC

proteins

We then asked how DHHC overproduction would affectsubstrates that are not exclusively localized to the vacuole.In previous studies, we used a fusion protein consistingof the N-terminal 18 amino acid sequence, termed Srchomology 4 (SH4) domain, of Vac8 and GFP (13,18).This SH4-GFP fusion protein is found on the vacuole andon the plasma membrane in wild-type cells (Figure 4A),but looses vacuole localization in the absence of Pfa3

(Figure 4C) as previously shown (13). A deletion in akr1did not affect the localization of SH4-GFP, but diminishedplasma membrane localization (Figure 4C). Moreover, thefusion protein was poorly localized to membranes ifseveral DHHCs were lacking (Figure 4B, right panel),indicating that it could be a substrate of multiple DHHCs.When we overproduced Erf2, Pfa4 and Pfa5 in wild-type or 5 × � cells, SH4-GFP accumulated at the plasmamembrane (Figure 4), and was palmitoylated. In contrast,overexpression of the vacuolar Pfa3 efficiently trappedSH4-GFP at the vacuole (Figure 4A,B, bottom panel). Onthe basis of these results, we speculate that palmitoylatedSH4-GFP traffics by default along the secretory pathwayto the plasma membrane. At the vacuole, Pfa3 canpalmitoylate and trap SH4-GFP due to the terminalcharacter of this organelle in the endomembrane system.

To further investigate the redundancy of DHHC proteins,we extended this analysis to other palmitoylated periph-eral membrane proteins, which – in part – had beenlinked to certain DHHC proteins (Figure 5A). The firstsubstrate, Yck3, contains several C-terminal cysteines,which have been shown to be modified by Pfa3 (13) andAkr1 (15,21). In fact, Yck3 is poorly localized (Figure 5B)and palmitoylated in the 5 × � mutant (15). When wetested Yck3 in our DHHC overexpression strains, weobserved clear vacuole localization in all cases (Figure 5B),which was not observed if the C-terminal cysteines werelacking (Figure S1A). This indicates that any DHHC canefficiently palmitoylate Yck3.

We then asked, how this DHHC-mediated palmitoylationconnects to the transport of Yck3, which occurs via the AP-3 pathway that connects Golgi and vacuole directly (21).In the absence of the AP-3 subunit Apl5, Yck3 is found atthe plasma membrane and – because of endocytosis – inthe lumen of the vacuole (Figure 5C). Overexpressionof selected DHHCs like the ER-localized Pfa4 or theplasma membrane DHHC Pfa5 did not restore vacuolar rimstaining (Figure 5C). In contrast, overproduction of Pfa3redirected Yck3 to the vacuole rim (Figure 5C, bottom).As a control, we show that Vac8 trafficking to the vacuoleis independent of an apl5 deletion (Figure S1B). Thus,overproduced Pfa3 can bypass the sorting information ofYck3, presumably by recruiting it directly from the cytosolto the vacuole surface.

As a second example, we analysed the casein kinaseYck2. Yck2 and its homolog Yck1 are dually palmitoylatedat their C-termini in an Akr1-dependent manner (10). Thesekinases are responsible for multiple phosphorylationevents at the plasma membrane and are mislocalizedif Akr1 is absent (22). As a consequence, akr1� cellshave an abnormal shape (15). We considered wild-type cell morphology as an indication for functionalrescue, when we screened our overexpressed DHHCproteins (Figure 5D). Whereas wild-type cells exhibiteda comparable shape in glucose and galactose, akr1�

cells were elongated in either medium. Overexpression

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Figure 3: Localization of Vac8 upon overexpression of DHHC proteins. (A–C) Localization of Vac8-GFP (A), Vac8 (C4,5,7A)-GFP (B)and Vac8 (G2A)-GFP (C) in the respective DHHC overexpression strains. Scale bar 10 μm. D) Subcellular fractionation was conductedas in Figure 1B. DHHC proteins were detected via the C-terminal Protein A tag. Pfa5 could not be detected in this experiment becauseof its low expression (see Figure 2A). E) Palmitoylation of wild-type Vac8 was determined by biotin switch as described in Methods.In brief, protein samples were pretreated with NEM to quench free cysteines, then reisolated and incubated with hydroxylamine tocleave the palmitate thioester. Free sulfhydryl groups were then cross-linked to BMCC biotin, and biotinylated proteins were enrichedon streptavidin beads. Load (4%) corresponds to the sample prior to the pull down.

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Figure 4: Targeting of SH4-GFP by DHHCs. Cells expressing genomically integrated SH4-GFP were analysed by fluorescencemicroscopy. Localization of SH4-GFP is shown in the wild-type (A) and 5 × � (B) background with different DHHCs overproduced. C)SH4-GFP localization in the indicated DHHC deletion strains. Size bar: 10 μm.

of all DHHCs, but the vacuole-localized Pfa3 rescuedthe morphology defect of akr1� cells (Figure 5D). Thissuggested that Yck1/2 palmitoylation can only occur ifthe DHHC proteins are localized along the secretorypathway. Alternatively, Pfa3 might be able to recognizeYck1/2, but would trap them at the wrong location. Todistinguish between these possibilities, we tagged Yck2with an N-terminal GFP tag to determine its localization.As previously described (10), Yck2 was mislocalizedto the cytosol in akr1� cells (Figure 5E). Akr1, Pfa4or Pfa5 overproduction directed Yck2 to the plasmamembrane (Figure 5E), consistent with the rescue inmorphology observed before (Figure 5D). In the Pfa4overexpression strain, some Yck2 was concentrated inone to two punctate structures, which interfered withvisualization of plasma membrane localization. Only forErf2 overexpression we observed a mixed result: cellmorphology was only rescued if Yck2 was not GFPtagged (Figure 5D,E). This could be because of thepoor recognition of Yck2 in the presence of the GFPtag or the slightly higher expression of Yck2. Moreimportantly, overexpression of Pfa3 led to a striking

vacuolar concentration of Yck2, indicating that Pfa3 isable to recognize Yck2, but traps it at the wrong location,where it cannot conduct its function (Figure 5D,E).

We selected the plasma membrane-localized Ras2-GTPase as a third example to analyse the responseto overexpressed DHHCs. Ras2 has a dual palmitoyland prenyl anchor at its C-terminus. The ER-localizedDHHC Erf2 has been identified as an acyltransferasefor Ras2. In the absence of Erf2, Ras2 is poorlypalmitoylated, but still found primarily at the plasmamembrane (8,23,24). In addition, GFP-Ras2 staining inthe cytosol is slightly increased, and a weak vacuolarring is visible (Figure 5F). Overexpression of Erf2, Akr1,Pfa4 and Pfa5 in the erf2 deletion background rescuedthe plasma membrane localization of Ras2. Only Pfa3overproduction redirected a significant portion of Ras2to the vacuole membrane in the erf2� background(Figure 5F). Interestingly, in the 5 × � cells (which stillhave Erf2) such a Pfa3-mediated redirection does notoccur (Figure S2). Potentially, Erf2 palmitoylation of Ras2 inwild-type cells occurs preferentially early in the secretory

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pathway, and the potential low turn over of Ras2 may notexpose it to Pfa3, even if it is overexpressed.

We conclude that DHHC proteins have a broad specificity,exemplified by the ability of Pfa3 to trap proteins at thevacuole.

Recognition of Vac8 by Pfa3

During our analysis of the substrate specificity of theDHHC proteins, we noticed that the dedicated enzyme-substrate pair Pfa3 and Vac8 behaved differently. Pfa3 wasable to trap even non-myristoylated or non-palmitoylated

Vac8 at vacuoles (Figure 3B,C). We therefore decided toanalyse this interaction in more detail. First, we generateda mutant form of Pfa3, in which the central cysteine (C134)of the DHHC motif was mutated to its non-functionalDHHA form (14,25). This mutation in Pfa3 did not affectits trafficking to the vacuole (Figure 2C,6A). Unexpectedly,the Pfa3 C134A rescued vacuolar localization of Vac8-GFP to a similar extent as wild-type Pfa3 (Figure 6B).We performed subcellular fractionation to confirm thelocalization of wild-type Vac8 (Figure 6C), and used theestablished biotin switch protocol (Figure 6D) to assessthe palmitoylation status of Vac8. Unlike wild-type Pfa3(Figure 6C, lanes 2 and 3), the C134A mutant is not able

Figure 5: Continued on next Page.

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Figure 5: Overlapping substrate specificity of DHHC proteins. A) Overview of DHHCs and corresponding substrates. Substratesand their acyltransferases as described in the literature are listed. The palmitoylation sites of each substrate are shown in bold.B) and C) Localization of GFP-Yck3 in the indicated strains was analysed by fluorescence microscopy as before. Size bar = 10 μm.D) Rescue of cell morphology by DHHC overexpression in akr1� cells. The indicated cells were grown in glucose (YPD) and galactose(YPG) and analysed by DIC optics. E) Localization of GFP-tagged Yck2 by fluorescence microscopy in cells overexpressing the indicatedDHHC proteins in the akr1� background. F) Localization of GFP-Ras2 in the presence or absence of the indicated overexpressed DHHCproteins in the erf2� background.

to efficiently retain Vac8 on vacuoles during subcellularfractionation (Figure 6C, lanes 11 and 12) or conferpalmitoylation (Figure 6D, lanes 7 and 8). This suggeststhat the myristoylated Vac8 is able to bind at leasttransiently to membranes and is retained at the vacuoleby directly binding to Pfa3 C134A in vivo. During thesubcellular fractionation procedure, this binding is probablyreduced because of the dramatic dilution. However, somewild-type Vac8 is still found on membranes and could beco-isolated with Pfa3 C134A, but not the wild-type Pfa3(Figure 6E, lanes 5 and 6), suggesting that Pfa3 C134Ais able to interact with non-palmitoylated Vac8 at thevacuole.

We analysed whether the Pfa3 C134A mutant couldinteract equally well with Vac8 lacking the N-terminalcysteines (Vac8 C4,5,7A) or the myristoylation site (G2A).As mentioned earlier, both mutant proteins localizeto vacuoles in the presence of overexpressed wild-type Pfa3 (Figure 3A–C). Interestingly, overexpression ofPfa3 C134A also conferred vacuole localization on Vac8C4,5,7A, but not the G2A mutant (Figure 6B), suggestingthat myristoylation is a prerequisite for the association ofVac8 with the enzymatically inactive Pfa3 C134A mutant.Indeed, neither mutant is palmitoylated (Figure 6F, lanes7 and 8). As observed for the Vac8 wild-type protein, theinteraction between the Pfa3 mutant and the Vac8 C4,5,7Awas not strong enough to completely withstand the

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Figure 6: Interaction of Pfa3 and Vac8. A) Localization of GFP-tagged Pfa3 (C134A) by fluorescence microscopy. Size bar = 10 μm. B)Localization of Vac8-GFP, Vac8 (C4,5,7A)-GFP and Vac8 (G2A)-GFP in cells overexpressing Pfa3 (C134A) was determined by fluorescencemicroscopy as previously described. C) Subcellular fractionation. The indicated cells carrying the Vac8-GFP wild-type or Vac8 with theC4,5,7A or G2A mutations in addition to endogenous Vac8 were grown in YPG to overexpress Pfa3 and subjected to subcellularfractionation (see Figure 1B and Methods). Proteins were analysed by SDS-PAGE and western blot using antibodies against the vacuolarV-ATPase subunit Vma6, Vac8 and Arc1. Pfa3 was detected via the C-terminal Protein A tag. D) Palmitoylation of wild-type Vac8 in thepresence of overexpressed Pfa3 and Pfa3-C134A was determined by biotin switch as described in Methods. E) Immunoprecipitationof Pfa3 and Pfa3 (C134A). C-terminal Protein A-tagged Pfa3 or Pfa3 (C134A) were immunoprecipitated as described in Methods, andtheir interaction with wild-type Vac8 was determined by SDS-PAGE and western blot using an antibody against Vac8. F) Palmitoylationof Vac8 (C4,5,7A)-GFP and Vac8 (G2A)-GFP in the presence of overexpressed Pfa3 and Pfa3-C134A was determined by biotin switchas described in Methods. G) Interaction of Pfa3 (C134A) with Vac8 mutant proteins. Cells with or without overexpressed Pfa3 (C134A),which expressed the indicated Vac8-GFP fusion protein, were subjected to immunoprecipitation and analysed as in (E).

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subcellular fractionation protocol (Figure 6C, lanes 14 and15), albeit some membrane-bound Vac8 C4,5,7A was alsoco-purified with Pfa3 C134A (Figure 6G, lanes 10 and 11).

We noticed that the non-myristoylated Vac8 G2A wascompletely cytosolic upon overexpression of enzymat-ically inactive Pfa3 (Figure 6B), but localized if wild-type Pfa3 was overproduced (Figure 3C). Indeed, Vac8G2A was efficiently palmitoylated under these condi-tions (Figure 6F, lane 6). This is not observed, if Pfa3is expressed at endogenous levels (18), suggesting thatincreasing the amount of Pfa3 and thus the acyltransferaseactivity on the vacuole can compensate for the inefficientmembrane binding of a non-myristoylated substrate pro-tein. One issue is, however, puzzling. Although the G2Amutant is efficiently palmitoylated upon Pfa3 overproduc-tion, a large fraction is found in the supernatant duringsubcellular fractionation (Figure 6C, lanes 8 and 9). It ispossible that either palmitoylation itself is not sufficient tosupport Vac8 G2A membrane localization or a portion ofVac8 G2A is released from membranes during the lysis byincreased depalmitoylation. When we analysed the pelletfraction, we could detect Vac8 G2A palmitoylation in themembrane fraction, which was comparable to the amountobserved in the total fraction (Figure S3). Thus, recruit-ment of Vac8 to Pfa3 is facilitated by its myristoyl anchor,but can be bypassed by higher expression levels of Pfa3. Inwild-type cells, this is not possible because of the 10-foldlower amount of Pfa3 compared to Vac8 (Figure 2B).

To obtain additional insight into the acyltransferase enzy-mology, we mutagenized Pfa3 and followed Vac8 palmi-toylation and localization. Initially, we made truncationmutants within the cytosolic DHHC domain. Any deletionwithin the DHHC domain inactivated Pfa3 (not shown).We therefore generated point mutants and followed theirconsequences on Vac8-GFP localization and palmitoyla-tion. To this end, we focused on amino acids (primarilycysteines) within the DHHC cytosolic domain, which werechanged individually or in pairs to alanine. We then fol-lowed Vac8 localization as a read-out. Mutagenesis ofconserved or non-conserved cysteines in combinationled to missorting of Vac8 to the cytosol (Figure 7A). Wethen focused on individual amino acids. Mutants in C106,C109 or C215 localized Vac8 to the vacuolar rim andpromoted palmitoylation (Figure 7A). However, cysteinesimmediately preceding the DHHC box (C120, C126) ormutating histidine residues (H118, H119) rendered Pfa3inactive for Vac8 palmitoylation (Figure 7A). Furthermore,we observed that mutations in the conserved DHHC boxhad distinct effects on Vac8 localization and palmitoy-lation. As shown above (Figure 6), the DHHA (C134A)mutant could localize Vac8 without palmitoylating theprotein, whereas a DHAC (H133A) mutant was inactive(Figure 7A). Surprisingly, a AHHC (D131A) mutant, whichwas expressed at a similar level in comparison to all othermutants analysed (not shown), was functional in bothassays, suggesting that the Asp residue is not essential

for palmitoylation. Our data therefore suggest that con-served residues preceding the DHHC box co-operate inthe palmitoylation reaction.

In summary, our data suggest a two-step acylationreaction, in which Pfa3 first recruits Vac8 by direct bind-ing, followed by the transfer of palmitate and subsequentrelease of the protein laterally to the membrane. Over-expression of the active acyltransferase can compensatefor poor initial membrane binding if the substrate lacks amyristoylation site. Owing to the loss of acyltransferaseactivity of Pfa3-DHHA, the enzyme-substrate complex isstabilized, leading to a prolonged residence time of Vac8on overproduced Pfa3.

Discussion

Broad specificity of DHHCs

Previous palmitoylation studies revealed the specificityof enzymes for substrate proteins: the yeast Pfa3 pro-tein promotes efficient Vac8 palmitoylation, and a subsetof human DHHC proteins including Hip14, mediate PSD-95, SNAP-25 and synaptotagmin palmitoylation (14,26,27).However, the redundancy of DHHC palmitoyl acyltrans-ferases has been implicated by several studies. Someproteins, such as yeast Vac8 or Ras2 were still par-tially localized even if the identified DHHC protein wasdeleted (13,24). Only a combination of several DHHC dele-tions led to a loss of palmitoylation of several proteins,including Meh1, Yck3 and Vac8 (15).

In this study, we show that five yeast DHHCscan palmitoylate multiple substrates if overexpressed.This observation may explain the compensation inpalmitoylation upon loss of one DHHC protein, even if theremaining DHHC proteins are present at their endogenousexpression level (15). In addition, we show that Pfa5 hasacyltransferase activity (Figures 3–5). The redundancy insubstrate recognition might be an important mechanismas proteins with a high turn over at membranes likethe mammalian Ras GTPase (28) could be regulatedat the level of DHHC activity. Our observation thatAkr1 can localize Ras2 is therefore consistent with aRas repalmitoylation at the mammalian Golgi (28). Thedepalmitoylation of Ras in mammalian cells could bebecause of a ubiquitous thioesterase or a relatively lowhalf-life of the thioester bond. So far, the Apt1 proteinhas been identified in yeast and mammalian cells asa Gpa1 (Gα)-specific thioesterase (29,30), but additionalstudies will be necessary to address its general functionin depalmitoylation.

Overexpression may bypass DHHC regulators, whichotherwise ensure palmitoylation by a selected DHHCprotein. In this respect, we were surprised to see rescueof activity if just Erf2, but not its co-factor Erf4 (8),was overexpressed. Potentially, a single Erf4 protein cansupport multiple Erf2 proteins.

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Figure 7: Mutagenesis of the DHHC cysteine-rich domain sequence. A) Overview of the Pfa3 mutants generated and theconsequences on localization. The marked cysteine (indicated by numbers), histidine and aspartate residues were changed individuallyor in pairs to alanine. For localization, Pfa3 was overexpressed as before in the Vac8-GFP background. Palmitoylation was determinedby the biotin switch protocol. B) Model of the interaction between Vac8 and Pfa3. Palmitate is indicated by the light gray color.

The vacuolar DHHC Pfa3 protein is unusual in that itcan mislocalize several proteins to the vacuole, includingRas2, Yck3, SH4-GFP and Yck2. We suggest that thisrecruitment occurs directly from the cytosol. In supportof this notion, we observed that Yck3, which is sortedvia the adapter protein complex (AP)-3 pathway to thevacuole (21) and can be modified by the ER-localized Pfa4(Figures 3,4), can reach the vacuole surface even if theAP-3 pathway has been disrupted (Figure 5).

This retention at the vacuole might be because of theslow turn over of palmitoylated proteins at the vacuole orthe fast repalmitoylation after hydrolysis of the thioesterbond. Why did we then not observe an accumulation

of substrates at the nuclear ER in cells overexpressingPfa4 or at the Golgi in Akr1 overexpressing cells? Evenbefore the DHHC family had been identified it waspostulated that the distribution of acyltransferases todifferent compartments is sufficient to kinetically trapsubstrates (31,32). Our data are consistent with a modelthat proteins, which become modified at the ER, Golgi orplasma membrane, enter secretory or endocytic vesicles,leave the organelles soon after their palmitoylation and getsorted based on their sorting information. Proteins lackingsorting information (SH4-GFP) or the sorting machinery(Yck3 in apl5�) seem to get palmitoylated at the ERand Golgi and will accumulate at the plasma membrane.Transport of some palmitoylated proteins to the plasma

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membrane may therefore not require a specific sortingsignal. Future studies using mutants that block secretionwill be necessary to test this model.

In our experiments, we use overexpression as a toolto show the versatility of DHHC proteins for theirsubstrates in yeast. However, in mammalian cells co-overexpression has been widely used to identify theDHHC and substrate pairs (26,27,33,34). Since we canshow that upon overproduction almost any DHHC testedcan recognize each analysed substrate, it should betaken into consideration that data from overexpressionapproaches that reveal specific enzyme-substrate pairsrequire a very careful interpretation.

Pfa3 provides insight into the palmitoylation

mechanism

Our analysis suggests at least two stages in the Pfa3-mediated acylation reaction: (i) binding of the substrateto the DHHC protein, followed by (ii) palmitoylation andlateral release into the membrane. In support of this, wefind that wild-type Pfa3 can recruit Vac8 lacking the N-terminal cysteines, and an enzymatically inactive DHHAvariant of Pfa3 mediates vacuole localization of wild-typeVac8 and a mutant lacking the N-terminal cysteines.The latter observation is consistent with our previousobservation, where we showed that a GFP-fusion proteincontaining the N-terminal 18 amino acids of Src, whichis positively charged but lacks cysteines, is localized tovacuoles in a Pfa3-dependent manner (13). In addition,the palmitoylation-independent recruitment of Pfa3 isdosage-dependent; Vac8 C4,5,7A membrane localizationis observed when Pfa3 is overproduced but not in wild-type cells (18), indicating that additional Pfa3 can recruitVac8 C4,5,7A to the vacuole even though it is only looselybound.

Recently, a palmitoylation-independent function has beensuggested for the DHHC protein Swf1 based on theanalysis of the corresponding DHHA mutant (35). As Pfa3-DHHA is able to localize Vac8 to vacuoles, we suggest thatSwf1 DHHA functions in a similar manner in its regulationof the actin cytoskeleton.

Several palmitoylated peripheral membrane proteinsrequire a second membrane-binding domain, such asmyristoylation (Vac8) and prenylation (Ras2). For Vac8,myristoylation is a prerequisite to promote an efficientinteraction with Pfa3. The poor recognition of non-myristolated Vac8 G2A can be bypassed by overproducingPfa3, which can bind and then palmitoylate Vac8 G2A(Figure 6F). Surprisingly, Vac8 G2A does not remainmembrane associated during subcellular fractionation.Our data suggest that the retention of the G2A mutantin vivo is primarily because of the interaction with andpalmitoylation by Pfa3.

Palmitoylation via Pfa3 requires the CRD proximal tothe DHHC box. Beside wild-type Pfa3, only Pfa3 with a

mutation of the active site cysteine is able to promote thelocalization of Vac8 to the vacuole membrane, whereasmutants within the CRD or a DHAC mutant, whichmaintain the central cysteine, inactivate Pfa3. Surprisingly,the conserved Asp residue of the DHHC box is notessential for palmitoylation activity of Pfa3, at least underthe conditions analysed. Similar mutations in Erf2 (Dto A) and Akr1 (DH to AA) caused inactivation of theprotein (10,23). Future experiments using purified proteinswill be necessary to analyse the catalytic mechanism indetail.

In summary, our analysis of Pfa3 and other DHHC pro-teins provides novel insight into the enzymatic mechanismby DHHC acyltransferase and their unexpected broad sub-strate specificity. We suggest that the broad distribution ofDHHC proteins along the endomembrane system couldprovide an acyltransferase network to ensure the pro-tein palmitoylation or repalmitoylation. Future studies willneed to address the acyltransferase mechanism and thepotential regulation of DHHC activity.

Methods

For yeast strains and molecular biology, see Supporting Information.

MicroscopyCells were centrifuged at 3000 × g and washed with phosphate bufferedsaline (PBS) buffer. Images of yeast cells were acquired with digital camera(SPOT pursuit XS monochrome; Diagnostic Instruments, Inc.) on a LEICADM5500B microscope equipped with differential interference contrast(DIC) optics and bandpass filter for GFP (37).

Biochemical proceduresCell lysates were prepared as described (18,36). Subcellular fractionationwas done as before (36,37). Pellet and supernatant were separated bycentrifugation at 20 000 × g for 10 min at 4◦C. For immunoprecipitations,cells were disrupted by glass beads in the PBS buffer (9 mM Na2HPO4,1.8 mM KH2PO4, 136 mM NaCl, 2.7 mM KCl, pH 6.8) with 0.5% Triton-X-100, 1× protease inhibitor cocktail (PIC; 0.1 μg/mL leupeptin, 1 mM

o-phenanthroline, 0.5 μg/mL pepstatin A, 0.1 mM pefablock) at 4◦C. Thelysate was clarified by centrifugation (20 000 × g, 10 min, 4◦C) and thesupernatant containing 2 mg protein in 400 μL was incubated with IgGsepharose beads (Sigma) for 2 h at 4◦C on a nutator. Beads were washedtwice and proteins were eluted by boiling in SDS sample buffer. They werethen analysed by SDS-PAGE and western blotting.

Biotin switch assayThe biotin switch assay was done as before (13,37) with slight modi-fication. After quenching of free cysteines by 25 mM n-ethylmaleimide(NEM) and following the chloroform/methanol precipitation, each sam-ple containing 1 mg protein was resuspended in 150 μL resuspen-sion buffer (2% SDS, 8 M urea, 100 mM NaCl, 50 mM Tris–HCl,pH 7.4) and incubated with 300 μM 1-Biotinamido-4-(4′[maleimidoethyl-cyclohexan]carboxamido)butane (BMCC-biotin) biotin BMCC in the pres-ence of 900 μL 1 M hydroxylamine, pH 7.4 on a turning wheel for 2 h at4◦C. Proteins were precipitated by chloroform/methanol and resuspendedin 100 μL resuspension buffer. The samples were diluted with 1 mL PBSbuffer containing 0.1% Triton-X-100, 5 mM EDTA, 0.1 PIC, and incubatedon a turning wheel for 15 min at 4◦C. Two or 4% of sample was savedas a loading control. The remaining sample was incubated with 30 μLneutravidin-agarose beads for 1 h at 4◦C on a turning wheel, washed twice

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Hou et al.

with PBS buffer containing 0.5 M NaCl, 0.1% Triton-X-100 and once withPBS. Proteins were eluted by boiling in SDS sample buffer and analysedby SDS-PAGE and western blotting.

Acknowledgments

We thank Nicolas Davis for yeast strains, Angela Perz for experimentalsupport, Clemens Ostrowicz for comments and all members of theUngermann lab for fruitful discussions. This work was supported by theSFB431. C.U. is supported by the Hans-Muhlenhoff foundation. A.T. JPwas supported by a mobility fellowship of the Boehringer IngelheimsFonds.

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1: Control for Yck3 sorting to vacuoles. A) Vacuole localizationof Yck3 depends on the C-terminal anchor. GFP-tagged Yck3 lackingthe cysteine-rich C-terminus (amino acid 517-525) was analysed in theindicated strains by fluorescence microscopy and DIC optics. B) Vac8-GFPlocalization in the indicated cells was analysed as in (A).

Figure S2: Localization of GFP-Ras2 in the presence of Erf2 cannot be

influenced by overexpressed Pfa3. Cells were analysed by fluorescencemicroscopy and DIC optics. 5 × � = akr1� akr2� pfa3� pfa4� pfa5�

Figure S3: Sorting of Vac8 and Yck3 upon overexpression of Pfa3-

C134A. A) Localization of GFP-Yck3 in cells overexpressing Pfa3-C134A.Analysis was as in Figure S1. B) Palmitoylation of Vac8 G2A-GFPon membrane fractions of cells overexpressing Pfa3. After subcellularfractionation, the pellet (P) was resuspended in the same amount of lysisbuffer as supernatant (S) and total lysate (T), and NEM was added in eachsample with 0.5% Triton-X-100. The following steps were the same asdescribed above. Labelling and pull down of biotinylated proteins was asdescribed in Methods and Figure 3E

Table S1: Strains used in this study.

Please note: Wiley-Blackwell are not responsible for the content or

functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

References

1. Hancock JF, Paterson H, Marshall CJ. A polybasic domain orpalmitoylation is required in addition to the CAAX motif to localizep21ras to the plasma membrane. Cell 1990;63:133–139.

2. Smotrys JE, Linder ME. Palmitoylation of intracellular signaling pro-teins: regulation and function. Annu Rev Biochem 2004;73:559–587.

3. Linder ME, Deschenes RJ. Palmitoylation: policing protein stabilityand traffic. Nat Rev Mol Cell Biol 2007;8:74–84.

4. Dietrich LE, Ungermann C. On the mechanism of protein palmitoyla-tion. EMBO Rep 2004;5:1053–1057.

5. Huang K, El-Husseini A. Modulation of neuronal protein trafficking andfunction by palmitoylation. Curr Opin Neurobiol 2005;15:527–535.

6. Linder ME, Deschenes RJ. Model organisms lead the way to proteinpalmitoyltransferases. J Cell Sci 2004;117:521–526.

7. Ohno Y, Kihara A, Sano T, Igarashi Y. Intracellular localizationand tissue-specific distribution of human and yeast DHHC

cysteine-rich domain-containing proteins. Biochim Biophys Acta2006;1761:474–483.

8. Lobo S, Greentree WK, Linder ME, Deschenes RJ. Identification of aRas palmitoyltransferase in Saccharomyces cerevisiae. J Biol Chem2002;277:41268–41273.

9. Valdez-Taubas J, Pelham H. Swf1-dependent palmitoylation of theSNARE Tlg1 prevents its ubiquitination and degradation. Embo J2005;24:2524–2532.

10. Roth AF, Feng Y, Chen L, Davis NG. The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. J Cell Biol2002;159:23–28.

11. Kihara A, Kurotsu F, Sano T, Iwaki S, Igarashi Y. Long-chain basekinase Lcb4 Is anchored to the membrane through its palmitoylationby Akr1. Mol Cell Biol 2005;25:9189–9197.

12. Lam KK, Davey M, Sun B, Roth AF, Davis NG, Conibear E. Palmitoy-lation by the DHHC protein Pfa4 regulates the ER exit of Chs3.J Cell Biol 2006;174:19–25.

13. Hou H, Subramanian K, LaGrassa TJ, Markgraf D, Dietrich LE,Urban J, Decker N, Ungermann C. The DHHC protein Pfa3 affectsvacuole-associated palmitoylation of the fusion factor Vac8. Proc NatlAcad Sci U S A 2005;102:17366–17371.

14. Smotrys JE, Schoenfish MJ, Stutz MA, Linder ME. The vacuolarDHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p.J Cell Biol 2005;170:1091–1099.

15. Roth AF, Wan J, Bailey AO, Sun B, Kuchar JA, Green WN, Phin-ney BS, Yates JR, 3rd, Davis NG. Global analysis of protein palmi-toylation in yeast. Cell 2006;125:1003–1013.

16. Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ,Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME,Phizicky EM, Snyder M, Grayhack EJ. Biochemical and geneticanalysis of the yeast proteome with a movable ORF collection.Genes Dev 2005;19:2816–2826.

17. Wang YX, Catlett NL, Weisman LS. Vac8p, a vacuolar protein witharmadillo repeats, functions in both vacuole inheritance and proteintargeting from the cytoplasm to vacuole. J Cell Biol 1998;140:1063–1074.

18. Subramanian K, Dietrich LE, Hou H, Lagrassa TJ, Meiringer CT,Ungermann C. Palmitoylation determines the function of Vac8 atthe yeast vacuole. J Cell Sci 2006;119:2477–2485.

19. Peng Y, Tang F, Weisman LS. Palmitoylation plays a role in targetingVac8p to specific membrane subdomains. Traffic 2006;7:1378–1387.

20. Pan X, Roberts P, Chen Y, Kvam E, Shulga N, Huang K, Lemmon S,Goldfarb DS. Nucleus-vacuole junctions in Saccharomyces cerevisiaeare formed through the direct interaction of Vac8p with Nvj1p.Mol Biol Cell 2000;11:2445–2457.

21. Sun B, Chen L, Cao W, Roth AF, Davis NG. The yeast casein kinaseYck3p is palmitoylated, then sorted to the vacuolar membranewith AP-3-dependent recognition of a YXXPhi adaptin sorting signal.Mol Biol Cell 2004;15:1397–1406.

22. Feng Y, Davis NG. Akr1p and the type I casein kinases act priorto the ubiquitination step of yeast endocytosis: Akr1p is requiredfor kinase localization to the plasma membrane. Mol Cell Biol 2000;20:5350–5359.

23. Bartels DJ, Mitchell DA, Dong X, Deschenes RJ. Erf2, a novel geneproduct that affects the localization and palmitoylation of Ras2 inSaccharomyces cerevisiae. Mol Cell Biol 1999;19:6775–6787.

24. Zhao L, Lobo S, Dong X, Ault AD, Deschenes RJ. Erf4p and Erf2pform an endoplasmic reticulum-associated complex involved in theplasma membrane localization of yeast Ras proteins. J Biol Chem2002;277:49352–49359.

25. Mitchell DA, Vasudevan A, Linder ME, Deschenes RJ. Protein palmi-toylation by a family of DHHC protein S-acyltransferases. J Lipid Res2006;47:1118–1127.

1072 Traffic 2009; 10: 1061–1073

Overlapping Substrate Specificity of DHHC Proteins

26. Huang K, Yanai A, Kang R, Arstikaitis P, Singaraja RR, Metzler M,Mullard A, Haigh B, Gauthier-Campbell C, Gutekunst CA, Hay-den MR, El-Husseini A. Huntingtin-interacting protein HIP14 is apalmitoyl transferase involved in palmitoylation and trafficking ofmultiple neuronal proteins. Neuron 2004;44:977–986.

27. Fukata M, Fukata Y, Adesnik H, Nicoll RA, Bredt DS. Identification ofPSD-95 palmitoylating enzymes. Neuron 2004;44:987–996.

28. Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C, Lumbierres M,Kuhlmann J, Waldmann H, Wittinghofer A, Bastiaens PI. An acylationcycle regulates localization and activity of palmitoylated Ras isoforms.Science 2005;307:1746–1752.

29. Yeh DC, Duncan JA, Yamashita S, Michel T. Depalmitoylation ofendothelial nitric-oxide synthase by acyl-protein thioesterase 1is potentiated by Ca(2+)-calmodulin. J Biol Chem 1999;274:33148–33154.

30. Duncan JA, Gilman AG. Characterization of Saccharomyces cere-visiae acyl-protein thioesterase 1, the enzyme responsible for Gprotein alpha subunit deacylation in vivo. J Biol Chem 2002;277:31740–31752.

31. Shahinian S, Silvius JR. Doubly-lipid-modified protein sequencemotifs exhibit long-lived anchorage to lipid bilayer membranes.Biochemistry 1995;34:3813–3822.

32. Nadolski MJ, Linder ME. Protein lipidation. Febs J 2007;274:5202–5210.

33. Fernandez-Hernando C, Fukata M, Bernatchez PN, Fukata Y, Lin MI,Bredt DS, Sessa WC. Identification of Golgi-localized acyltransferases that palmitoylate and regulate endothelial nitric oxidesynthase. J Cell Biol 2006;174:369–377.

34. Fang C, Deng L, Keller CA, Fukata M, Fukata Y, Chen G, Luscher B.GODZ-mediated palmitoylation of GABA(A) receptors is required fornormal assembly and function of GABAergic inhibitory synapses.J Neurosci 2006;26:12758–12768.

35. Dighe SA, Kozminski KG. Swf1p, a member of the DHHC-CRDfamily of palmitoyltransferases, regulates the actin cytoskeleton andpolarized secretion independently of its DHHC motif. Mol Biol Cell2008;19:4454–4468.

36. Lagrassa TJ, Ungermann C. The vacuolar kinase Yck3 maintainsorganelle fragmentation by regulating the HOPS tethering complex.J Cell Biol 2005;168:401–414.

37. Meiringer CT, Auffarth K, Hou H, Ungermann C. Depalmitoylation ofYkt6 prevents its entry into the multivesicular body pathway. Traffic2008;9:1510–1521.

Traffic 2009; 10: 1061–1073 1073