Our search for trafficking proteins that specifically associate with the γ2 subunit has identified GODZ as a palmitoyltransferase of GABAA receptors. In HEK 293T cells, palmitoylation of the γ2 subunit is dependent on cotransfection of GODZ. Palmitoylation of the γ2 subunit was similarly detected in neurons. Moreover, the regional and neuron-specific expression pattern of GODZ in brain is reminiscent of that of the γ2 subunit. The data are consistent with GODZ being involved in palmitoylation of GABAA receptors in vivo.
The γ2 subunit contains five cysteine residues in the putative cytoplasmic loop region that are conserved in all three γ subunits and might function as palmitoylation sites. Four of these cysteines map to the minimal GODZ interaction site, which is highly conserved in all three γ subunits. In contrast, the putative cytoplasmic domains of α, β, δ, and π subunits are devoid of cysteine residues, suggesting that palmitoylation represents a posttranslational modification that is specific for GABAA receptor subtypes that are preferentially found at postsynaptic sites. The cytoplasmic domains of ε, θ, and ρ subunits contain between two and six cysteine residues; however, the position and flanking sequences are different from those in the γ subunits, and their subcellular distribution has not been analyzed.
Preliminary analyses of γ
2 subunit constructs containing single mutated cysteines indicate that GODZ-mediated palmitoylation of the γ
2 subunit occurs at more than one residue (C. A. Keller and B. Lüscher, unpublished observations). Detailed mapping of palmitoylation sites is complicated, however, by the notion that substitution of cysteine residues by other amino acids can affect interaction of GODZ with its substrate even if the cysteines are not normally palmitoylated. Reduced palmitoylation of a construct with mutated cysteine residues is therefore not sufficient to unambiguously identify the palmitoylated residue(s). However, while this manuscript was under review, we learned of a report by Rathenberg et al. (2004)
who also demonstrated palmitoylation of the γ
2 subunit in neurons. In addition, they showed that cysteine to alanine substitutions in the cytoplasmic loop region of the γ
2 subunit interfere with trafficking of this subunit to the plasma membrane of transfected neurons and, probably as a consequence, to a deficit in postsynaptic clustering. Together the data suggest that palmitoylation of the γ
2 subunit by GODZ might play a role in membrane trafficking of postsynaptic GABAA
The first identification of DHHC-CRD proteins as palmitoyltransferases is based on genetic and biochemical analyses of the yeast proteins Akr1 and Erf2p/Erf4p. Mutations in the DHHC-CRD domains in Akr1 and Erf2 disrupt palmitoylation by these proteins (Bartels et al., 1999
; Lobo et al., 2002
; Roth et al., 2002
). Similarly, in-frame deletion of the center portion of GODZ containing the DHHC-CRD domain abolished interaction with the γ
2 subunit and suggests that the DHHC-CRD domain serves as a signature feature of a diverse class of palmitoyltransferases. Similar to Erf2 and Akr1, GODZ is subject to self-palmitoylation, suggesting this is a general feature of this class of enzymes.
Attempts at biochemical characterization and cloning of palmitoyltransferases has been hampered by their integral association with membranes and because of their inherent instability in vitro
(Berthiaume and Resh, 1995
; Dunphy et al., 1996
). In agreement, native and recombinant GODZ was found to copurify with membranes. Furthermore, immunocomplexes of GODZ and GABAA
receptors exhibited an unusually short half-life of only a couple of hours in vitro
, and not surprisingly, GODZ was unable to associate with purified γ
2 subunit bait proteins in pull-down assays (data not shown). Thus, the association seen between GODZ and GABAA
receptors likely reflects the transient interaction of a typical enzyme with one of its substrates. Interaction of GODZ with the γ
2 subunit in the SOS recruitment–yeast two-hybrid system was dependent on the functional integrity of all four putative transmembrane domains of GODZ, which suggests that this interaction could not be detected by a standard yeast two-hybrid system that requires interaction of bait and prey proteins in the nucleus.
GODZ immunoreactivity is confined to the Golgi complex, where GABAA
receptors do not normally accumulate. Not surprisingly, no colocalization of GODZ and GABAA
receptors was detected in neurons, and no stable complex could be immunoprecipitated from neuron or brain extracts. In contrast, significant colocalization was evident, and stable immunocomplexes were isolated from transfected HEK 293T cells, suggesting the accumulation of an unusually long-lived reaction intermediate in these cells. Interestingly, the yeast palmitoyltransferase Erf2p copurifies with a second factor Erf4p that is part of the same complex and also essential for palmitoyltransferase activity, although it does not exhibit enzyme activity on its own and lacks the DHHC signature sequence (Lobo et al., 2002
). By analogy, we hypothesize that GODZ exists in a complex with another so far unidentified subunit that is essential for maximal processivity of this enzyme. In the absence of this other protein (i.e., in HEK 293T cells), the reaction might proceed more slowly, allowing detection of a reaction intermediate that does not normally accumulate, such as the complex between GODZ and the γ
2 subunit observed in HEK 293T cells. In agreement, no GABAA
receptor immunoreactivity is normally detected in the Golgi complex of neurons, although these receptors must traffic through the Golgi before reaching the plasma membrane.
Palmitoylation represents a novel posttranslational modification of GABAA
receptors and is known as a reversible modification involved in regulated trafficking and functional modulation of diverse proteins, especially in neurons (for review, see El-Husseini and Bredt, 2002
; Patterson, 2002
; Bijlmakers and Marsh, 2003
; Linder and Deschenes, 2003
). Neural proteins that are subject to palmitoylation are structurally and functionally diverse and include both peripheral membrane-associated proteins that depend on lipid modification for membrane association and bona fide integral membrane proteins with one or several transmembrane domains. Similar to the γ
2 subunit, other multipass transmembrane proteins are typically palmitoylated at cysteines in proximity to the last transmembrane domain. Examples include diverse G-protein-coupled receptors and the GluR6 subunit of kainite receptors (O'Dowd et al., 1989
; Papac et al., 1992
; Pickering et al., 1995
; Moffett et al., 1996
; for review, see Qanbar and Bouvier, 2003
). Single-pass transmembrane proteins such as the synaptic vesicle proteins synaptotagmin (Chapman et al., 1996
) and synaptobrevin (Gonzalo et al., 1999
) are similarly palmitoylated in the cytosolic domain near the transmembrane domain. These proteins do not reveal an obvious consensus sequence for palmitoylation, however, suggesting that they are palmitoylated by distinct members of the DHHC family of palmitoyltransferases.
The mammalian genome contains at least 23 members of the DHHC family of proteins. GODZ is representative of a small subfamily of these DHHC proteins that contain four putative transmembrane regions and relatively short N- and C-terminal extensions, similar to Erf2p (Li et al., 2002
; Lobo et al., 2002
). Yeast two-hybrid assays indicate that only two of the four GODZ paralogs interact with the γ
subunits in yeast two-hybrid tests. This finding might indicate that other than GODZ, only ZDHHC7 can thioacylate the γ
2 subunit. Nevertheless, it cannot be excluded that γ
subunits are substrates for palmitoylation by several DHHC proteins, including SERZ-β
or other DHHC-CRD proteins that are more distantly related to GODZ. In addition, the degree of palmitoylation is likely to be regulated by specific palmitoylthioesterases. A first cytosolic enzyme implicated in noncatabolic deacylation of a subset of palmitoylated proteins is known as acylprotein thioesterase 1 (APT1) (Sugimoto et al., 1996
) or lysophospholipase I (Lyso PLA I) (Wang et al., 1997
). Two additional protein palmitoylthioesterases (PPT1/2) involved in lysosomal and therefore catabolic depalmitoylation have also been described (Camp et al., 1994
; Soyombo and Hofmann, 1997
; for review, see Linder and Deschenes, 2003
The different GODZ orthologs exhibit highest sequence similarity in the putative cytoplasmic region that contains the DHHC-CRD domain (96.3% similarity between GODZ and ZDHHC7; 58.8% similarity between GODZ and ZDHHC21) but only low homology at their N and C termini (47.7 and 53.0% similarity of N and C termini, respectively, between GODZ and ZDHHC7; 13.6 and 39.4% similarity of N and C termini, respectively, between GODZ and ZDHHC21). The putative transmembrane domains show intermediate levels of conservation (). Thus, although the central domain including the DHHC-CRD domain is highly conserved among GODZ and its paralogs and essential for interaction with the γ2 subunit, the variable N and C termini are likely to influence subcellular localization or substrate selectivity, or both.
The data presented here further support the pivotal role of γ subunits in trafficking of GABAA receptors. On the basis of the preferential localization of GODZ to one face of the Golgi, we favor a model whereby GODZ-mediated palmitoylation contributes to exocytosis of GABAA receptors or determines the stability and mobility of receptors in the membrane, rather than endoplasmic reticulum (ER) to Golgi or intra-Golgi transport. Consistent with this interpretation, no colocalization was detected between ER-resident marker proteins and GODZ in primary cultured neurons (data not shown). We speculate that GODZ-mediated palmitoylation contributes to sorting of GABAA receptors to distinct trafficking vesicles, possibly in concert with other vesicle-specific factors; however, a role for GODZ in endocytic trafficking and recycling of γ subunit-containing receptors can currently not be excluded. Future experiments will help to resolve these issues.