Jennifer Greaves

Functional and spatial segregation of secretory vesicle pools according to vesicle age

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

DHHC palmitoyl transferases : substrate interactions and (patho)physiology

S-palmitoylation is a reversible post-translational modification that occurs on diverse cellular proteins. Palmitoylation can affect proteins in many different ways, including regulating membrane attachment, intracellular trafficking, and membrane micro-localisation. Intracellular palmitoylation reactions are mediated by a family of recently identified aspartate-histidine-histidine-cysteine (DHHC) palmitoyl transferases. More than 20 DHHC proteins are encoded by mammalian genomes, and there is now a major effort to identify DHHC-substrate pairings and to determine how interaction specificity is encoded. Recent studies have highlighted how DHHC proteins regulate cell function and influence physiology and pathophysiology.

The intracellular dynamic of protein palmitoylation

S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation.

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming α-subunits. However, the molecular mechanism(s) by which phosphorylation of the α-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single α-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

Palmitoylation and Membrane Interactions of the Neuroprotective Chaperone Cysteine-string Protein

Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane “sampling.” Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.

Palmitoylation of the SNAP25 Protein Family SPECIFICITY AND REGULATION BY DHHC PALMITOYL TRANSFERASES

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.

The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases

SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre‐synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF (N‐ethylmaleimide‐sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre‐synaptic plasma membrane and vesicle‐associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca2+ levels, and a major Ca2+ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca2+ entry to vesicle fusion via Ca2+‐dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.

The palmitoyl transferase DHHC2 targets a dynamic membrane cycling pathway : regulation by a C-terminal domain

DHHC2 regulates dynamic palmitoylation of PSD95, a process that appears to be linked to changes in DHHC2 localization. The precise distribution and membrane dynamics of DHHC2 are not clear. We demonstrate that DHHC2 cycles between the plasma membrane and recycling endosomes (RE) and that this dynamic localization requires an intact C-terminal domain.