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Several members of the Src family of non-receptor protein tyrosine kinases (e.g. Yes), as well as Leishmania hydrophilic acylated surface protein B (HASPB) harbor a short N-terminal motif called the Src homology 4 (SH4) domain, which undergoes tandem modification with the saturated acyl chains myristate and palmitate. SH4 domains are responsible for stable anchoring of these otherwise soluble proteins to the cytoplasmic leaflet of cellular membranes, mediate their targeting to the plasma membrane, and moreover, confer affinity for cholesterol- and sphingolipid-enriched membrane microdomains, that is lipid rafts. N-terminal myristoylation occurs in the cytosol, concurrently with translation of the protein, and is a prerequisite for subsequent palmitoylation. The latter is thought to occur at perinuclear (most probably Golgi) membranes. It has been hypothesized that doubly acylated SH4 domain proteins partition into lipid rafts soon after being palmitoylated at the Golgi, and that these microdomains are necessary for the transport of SH4 proteins to the plasma membrane, by playing a role in their sorting and/or formation of transport carriers at the trans-Golgi network.
The aim of the first part of this study was to characterize membrane (both lipid and protein) environment of diacylated SH4 domain proteins residing in lipid rafts. The approach was to analyze lipid and protein components of immunoaffinity-purified detergent-resistant membranes (DRMs) containing SH4 domain reporter fusion proteins. DRMs immunoisolated using a HASPB SH4 domain fusion protein as bait differed in lipid composition from the total DRM pool, being enriched in sphingomyelin, and depleted in phosphatidylcholine and phosphatidylethanolamine, and therefore seem to be a subset of total DRMs. This may suggest that in vivo, the protein associates with a specific subset of lipid rafts, thus indicating that the heterogeneity of lipid rafts can be appreciated also using the detergent method. Our immunoaffinity purification approach seemed to enrich for bona fide raft proteins, as suggested by an increase compared to total DRMs in the proportion of plasma membrane and lipid-anchored proteins, as well as proteins whose association with DRMs is sensitive to cholesterol depletion. We estimated relative amounts of proteins in immunoisolated SH4 DRMs and total DRMs, with the use of label-free mass spectrometry-based quantification, which was validated for a subset of proteins by quantitative Western blotting. We could observe a lack of enrichment of endogenous Yes kinase in immunoisolated SH4 DRMs, which may indicate that the identity of lipid rafts into which SH4 domain proteins partition, is determined not only by the dual fatty acylation with myristate and palmitate, but also by interactions conferred by domains distal from the SH4 domain.
In the second part of this study, we intended to investigate the previously reported role of the COPI coatomer complex, as well as of the secretory pathway in general, in the plasma membrane transport of diacylated SH4 domain proteins. Conditions that disrupted the structure and function of the Golgi apparatus, i.e. siRNA-mediated knockdown of the β subunit of the COPI complex, expression of constitutively active mutant (Q71L) of the small GTPase Arf1, as well as brefeldin A treatment, resulted in increased intracellular accumulation of a HASPB SH4 fluorescent fusion protein, pointing out to the role of the Golgi in its transport to the plasma membrane. None of the above treatments, however, blocked the appearance of the SH4 reporter protein at the plasma membrane, suggesting the existence of an alternative trafficking pathway that does not require the Golgi complex.
|Supervisor:||Nickel, Prof. Dr. Walter|
|Date of thesis defense:||6 June 2013|
|Date Deposited:||11 Jun 2013 07:58|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|