Dissociation of Golgi-associated DHHC-type Zinc Finger Protein (GODZ)- and Sertoli Cell Gene with a Zinc Finger Domain-β (SERZ-β)-mediated Palmitoylation by Loss of Function Analyses in Knock-out Mice

Abstract

Edited by F. Anne StephensonThe ␥2 subunit of GABA type A receptors (GABA A Rs) is thought to be subject to palmitoylation by both Golgi-associated DHHC-type zinc finger protein (GODZ; also known as DHHC3) and its paralog Sertoli cell gene with a zinc finger domain-␤ (SERZ-␤; DHHC7) based on overexpression of enzymes and substrates in heterologous cells. Here we have further investigated the substrate specificity of these enzymes by characterization of GODZ and SERZ-␤ knock-out (KO) mice as well as double KO (DKO) neurons. Palmitoylation of ␥2 and a second substrate, growth-associated protein of 43 kDa, that is independently implicated in trafficking of GABA A Rs was significantly reduced in brain of GODZ KO versus wild-type (WT) mice but unaltered in SERZ-␤ KO mice. Accumulation of GABA A Rs at synapses, GABAergic innervation, and synaptic function were reduced in GODZ KO and DKO neurons to a similar extent, indicating that SERZ-␤ does not contribute to palmitoylation or trafficking of GABA A Rs even in the absence of GODZ. Notably, these effects were seen only when mutant neurons were grown in competition with WT neurons, thereby mimicking conditions of shRNA-transfected neurons previously used to characterize GODZ. However, GABA-evoked whole-cell currents of DKO neurons and the GABA A R cell surface expression in DKO neurons and GODZ or SERZ-␤ KO brain slices were unaltered, indicating that GODZ-mediated palmitoylation selectively controls the pool of receptors at synapses. The different substrate specificities of GODZ and SERZ-␤ in vivo were correlated with their differential localization to cis-versus trans-Golgi compartment, a mechanism that was compromised by overexpression of GODZ.S-Palmitoylation is an important posttranslational modification that involves the addition of the 16-carbon fatty acid chain palmitate via a thioester bond to Cys residues (1). This modification in turn can alter a protein's conformational state, membrane association, and complex formation as well as its susceptibility to other posttranslational modifications (2-4). Global analyses of rat synaptosomal fractions led to the discovery of nearly 300 palmitoylated synaptosomal protein candidates that illustrate the particular importance of palmitoylation in regulating the function of neuronal synapses (5).In mammalian cells, the palmitoylation reaction is catalyzed principally by a super gene family of 23 palmitoyl acyltransferases containing a DHHC motif in a cysteine-rich domain (DHHC-CRD) 3 that are both essential for enzyme function (6 -10). The mechanisms that determine substrate specificity of PATs remain poorly understood, although some specificity is observed upon overexpression of substrates and PATs in heterologous cells. However, increasing evidence suggests that the substrate specificity of DHHC-type PATs in vivo is much more stringent than in vitro. For example, the postsynaptic density (PSD) protein of 95-kDa (PSD-95) can be palmitoylated in heterologous cells by at least five members of the DHHC family of PATs (11), w...

DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels

“…Because DHHC3 and DHHC7 belong to the same subfamily of PATs with high sequence homology within the CRD (25), it is not surprising that these enzymes may have overlapping substrates. On the other hand, in vivo studies have revealed distinct substrate specificity for these PATs in the mouse brain (35). Knockdown experiments in cultured cells demonstrated that the presence of endogenous DHHC3 is not enough to fully compensate for DHHC7 deficiency.…”

The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection

“…Analysis of the roles of UL20 and its regulation in vivo are hampered by the essential role of UL20 and the inability of UL20 deletion mutants of HSV-1 to replicate efficiently in vitro or in vivo (3,4,32). Our recent construction of GODZ Ϫ/Ϫ mice (19), however, enabled investigation of the contribution of the GODZ component of the UL20-gK network to HSV-1 infectivity using MEFs derived from these mice, as well as in vivo analysis. In the current study, we demonstrate that HSV-1 replication in MEFs derived from GODZ Ϫ/Ϫ mice is markedly reduced compared with replication in MEFs from control WT mice.…”

“…We recently generated a mouse strain lacking GODZ expression (19). Characterization of these GODZ Ϫ/Ϫ mice confirmed that palmitoylation of the ␥2 subunit of GABA A receptors and of a second substrate, a growth-associated protein of 43 kDa that is involved in trafficking of GABA A receptors, was significantly reduced in the brains of ( Fig.…”

“…GODZ is localized predominately within the cis-Golgi complex (12,18,19) and has been shown to be involved in the palmitoylation of several proteins, especially in neurons (18)(19)(20)(21)(22). Using a dominant-negative construct of GODZ to characterize its role in virus infectivity in vitro, we have shown that the interaction of UL20 with GODZ is required for optimal virus infectivity, that UL20 is palmitoylated by GODZ in cultured cells, and that this palmitoylation is required for virus infectivity (6).…”

“…It is well established that GODZ is a Golgi complex protein (12,18,19), and binding of GODZ with UL20 has been shown to occur within the Golgi complex (6). We therefore infected MEFs from WT and GODZ Ϫ/Ϫ mice with VC1 virus for 16 h and then fixed and permeabilized the infected cells prior to incubation with anti-GM130 (to detect the Golgi complex), as well as anti-FLAG (to detect UL20) and anti-V5 (to detect gK) antibodies ( Fig.…”