ABSTRACT
It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a ß strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.
Subject(s)
Cytoplasm/metabolism , Cytoplasmic Dyneins/metabolism , Dyneins/metabolism , Microtubules/metabolism , Models, Molecular , Monomeric GTP-Binding Proteins/metabolism , rab3 GTP-Binding Proteins/metabolism , Amino Acid Substitution , Animals , COS Cells , Chlorocebus aethiops , Cytoplasm/enzymology , Cytoplasmic Dyneins/chemistry , Cytoplasmic Dyneins/genetics , Dimerization , Dyneins/chemistry , Dyneins/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Microtubules/enzymology , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/genetics , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Transport , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , rab3 GTP-Binding Proteins/chemistry , rab3 GTP-Binding Proteins/geneticsABSTRACT
Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16-carbon fatty acid palmitate, is the most common acylation of proteins in eukaryotic cells. This post-translational modification provides an important mechanism for regulating protein subcellular localization, stability, trafficking, translocation to lipid rafts, aggregation, interaction with effectors and other aspects of protein function. In addition, N-terminal myristoylation and C-terminal prenylation, two well-studied post-translational modifications, frequently precede protein S-palmitoylation at a nearby spot of the polypeptide chain. Whereas N-myristoylation and prenylation are considered essentially irreversible attachments, S-palmitoylation is a tightly regulated, reversible modification. In addition, the unique reversibility of protein palmitoylation also allows proteins to rapidly shuttle between intracellular membrane compartments in a process controlled, in some cases, by the DHHC family of palmitoyl transferases. Recent cotransfection experiments using the DHHC family of protein palmitoyl transferases as well as RNA interference results have revealed that these enzymes, frequently localized to the Golgi apparatus, tightly control subcellular trafficking of acylated proteins. In this article we will give an overview of how protein palmitoylation regulates protein trafficking and subcellular localization.
