S-palmitoylation is the reversible post-translational attachment of the 16-carbon saturated fatty acid palmitate to the sulphydryl group of cysteine residues through a thioester linkage. The primary function of palmitoylation has often been regarded as simply a means to increase the hydrophobicity and membrane affinity of modified proteins, allowing them to become stably membrane-associated. However, recent evidence has demonstrated additional roles for palmitoylation in regulating protein trafficking, the membrane micro-localisation of proteins, protein stability, and protein-protein interactions. A family of twenty-three mammalian proteins containing a conserved DHHC cysteine-rich domain have recently been identified, and several of these proteins have been shown to palmitoylate specific substrate proteins. The DHHC family of palmitoyl transferases are polytopic membrane proteins containing 4-6 membrane-spanning domains that are localised to distinct intracellular membranes, including the endoplasmic reticulum, Golgi apparatus, endosomes, and the plasma membrane. Since palmitoyl transferases are integral membrane proteins, substrate proteins must contain additional membrane targeting signals that mediate membrane association prior to palmitate transfer. For many proteins, the mechanism that mediates initial membrane binding prior to palmitoylation is clear; for example, initial membrane binding can be mediated by transmembrane domains or other lipid modifications such as prenyl or myristoyl groups, which are added to proteins in the cytosol. However, a number of palmitoylated proteins lack any obvious membrane targeting motifs and it is unclear how this class of proteins associate with membranes to allow palmitoylation to occur. The vesicle-associated, exocytotic chaperone Cysteine String Protein (CSP) is an example of such a protein. CSP is extensively palmitoylated on a "string' of fourteen cysteine residues present within its signature cysteine string domain. Palmitoylation of CSP is essential for its intracellular sorting and function, and thus it is important to understand how this essential modification of CSP is regulated. In this study, a detailed mutagenesis approach has been employed to elucidate the mechanisms governing the initial membrane targeting of CSP, and the enzymes that palmitoylate CSP have been identified. As well as providing important information on CSP palmitoylation and membrane interactions, it is hoped that this analysis will also serve as a paradigm to understand the mechanisms by which other proteins become palmitoylated. As a first step to characterising CSP membrane interactions, a hydrophobic 31 amino acid domain of CSP was identified as the minimal membrane binding domain present within the protein. This domain includes the cysteine string domain, and indeed cysteine residues within this domain are proposed to play an essential role in membrane interaction prior to palmitoylation. Membrane association of the minimal membrane binding domain is not sufficient to trigger palmitoylation, which requires additional residues downstream of the cysteine string domain. Intriguingly one role of these downstream residues in CSP appears to be to weaken membrane affinity and indeed, in contrast to the minimal membrane binding domain, full-length CSP was cytosolic in the absence of palmitoylation. The family of 23 DHHC proteins were screened for activity against CSP, showing that palmitoylation is specifically enhanced by co-expression of the Golgi-localised palmitoyl transferases DHHC3, DHHC7, DHHC15 or DHHC17; co-expression of these enzymes is sufficient to promote the stable membrane attachment of CSP. CSP mutants with an increased membrane affinity were localised to the ER, and thus physically separated from the Golgi-localised partner DHHC proteins of CSP, offering an explanation of why these mutants are not palmitoylated. Interestingly, palmitoylation of an ER-localised mutant could be rescued by Brefeldin A (BFA) treatment, which promotes the mixing of ER and Golgi membranes, supporting the view that mutants with an increased membrane affinity are not palmitoylated because they associate with 'inappropriate' membranes. In addition, the palmitoylated mutant remained at the ER following BFA washout and did not traffic to more distal membrane compartments, suggesting that palmitoylation of CSP may have a specific requirement to take place at Golgi membranes to facilitate subsequent intracellular sorting. A model is proposed whereby CSP utilises a weak membrane affinity to "sample" intracellular membranes for DHHC protein content. As partner DHHC proteins of CSP are restricted to the Golgi, palmitoylation and stable membrane attachment only occurs at this intracellular compartment. Mutations that enhance initial membrane affinity prevent sampling and lead to accumulation of CSP on abundant cellular membranes such as the ER. As a palmitoylated CSP mutant did not traffic from the ER, the coupling of CSP palmitoylation to Golgi membranes may therefore be an important requirement for subsequent sorting. These findings suggest that membrane "sampling" through specialised protein domains might be a common mechanism employed by substrate proteins to locate their specific DHHC partner proteins