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Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras, N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.

<p>Protein folding and export to the membrane are crucial events in the cell. Both processes may be initiated already at the ribosome, assisted by factors that bind to the polypeptide as it emerges from the ribosome. The signal recognition particle (SRP) scans the ribosome for nascent peptides destined for membrane insertion and targets these ribosomes to the site for translocation in the membrane. Trigger factor (TF) is a folding chaperone that interacts with nascent chains to promote their correct folding, prevent misfolding and aggregation. </p><p>In this thesis, we first investigated membrane targeting and insertion of two heterologous membrane proteins in E. coli by using in vitro translation, membrane targeting and cross-linking. We found that these proteins are dependent on SRP for targeting and that they initially interact with translocon components in the same way as native nascent membrane proteins. </p><p>Moreover we have characterised the SRP and TF interactions with the ribosome both with cross-linking experiments and with quantitative binding experiments. Both SRP and TF bind to ribosomal L23 close to the nascent peptide exit site where they are strategically placed for binding to the nascent polypeptide. </p><p>Quantitative analysis of TF and SRP binding determined their respective KD values for binding to non translating ribosomes and reveals that they bind simultaneously to the ribosome, thus having separate binding sites on L23. </p><p>Finally, binding studies on ribosome nascent chain adds clues as to how TF functions as a chaperone.</p>

Protein folding and export to the membrane are crucial events in the cell. Both processes may be initiated already at the ribosome, assisted by factors that bind to the polypeptide as it emerges from the ribosome. The signal recognition particle (SRP) scans the ribosome for nascent peptides destined for membrane insertion and targets these ribosomes to the site for translocation in the membrane. Trigger factor (TF) is a folding chaperone that interacts with nascent chains to promote their correct folding, prevent misfolding and aggregation. In this thesis, we first investigated membrane targeting and insertion of two heterologous membrane proteins in E. coli by using in vitro translation, membrane targeting and cross-linking. We found that these proteins are dependent on SRP for targeting and that they initially interact with translocon components in the same way as native nascent membrane proteins. Moreover we have characterised the SRP and TF interactions with the ribosome both with cross-linking experiments and with quantitative binding experiments. Both SRP and TF bind to ribosomal L23 close to the nascent peptide exit site where they are strategically placed for binding to the nascent polypeptide. Quantitative analysis of TF and SRP binding determined their respective KD values for binding to non translating ribosomes and reveals that they bind simultaneously to the ribosome, thus having separate binding sites on L23. Finally, binding studies on ribosome nascent chain adds clues as to how TF functions as a chaperone.