The on–off story of protein palmitoylation Bijlmakers, Marie-José; Marsh, Mark
Trends in cell biology,
2003, 2003-Jan, 2003-01-00, 20030101, Letnik:
13, Številka:
1
Journal Article
Recenzirano
Palmitoylation is one of the most frequent post-translational modifications found on proteins. It contributes to membrane association, protein sorting and many other processes. Through its ...reversibility, palmitoylation also provides mechanisms to regulate the functional activities of integral and peripheral membrane proteins. Here we discuss evidence that proteins can be palmitoylated at different locations in the cell, how targeting to these locations might be directed, and aspects of the proposed functions of palmitoylation.
The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, ...yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.
The MHC class II-associated invariant chain interacts in trimeric form with class II molecules, inhibits peptide binding, and mediates targeting of class II molecules to endosomal compartments. To ...dissect the different functions of the invariant (Ii) chain, a set of cDNAs, encoding truncated forms of the Ii chain, was constructed. mRNAs, transcribed from these cDNAs were translated in vitro, together with mRNAs encoding class II HLA DR1 alpha and beta subunits. An Ii chain truncation that contains the 104 NH2-terminal amino acids was able to associate with class II molecules. This construct contains the region from which class II-associated Ii chain peptides (CLIP, amino acids 81-104) are derived. The absence of a further eight residues at the COOH terminus results in a construct of 96 amino acids that is unable to associate with class II molecules. Association of the truncated Ii chains with class II molecules showed a strict correlation with inhibition of peptide binding. Removal of the NH2-terminal cytoplasmic tail and transmembrane region of Ii chain and its replacement with a cleavable signal sequence led to aberrant folding and impaired association with class II molecules. The region between amino acids 163 and 183 was found to be essential for visualization of Ii chain homotrimers by covalent cross-linking.
Human CMV (HCMV) infection leads to an almost complete inhibition of expression of MHC class I proteins. After infection with HCMV, the biosynthesis of HLA class I molecules was examined in human ...lung fibroblasts and in mouse fibroblasts transfected with genes encoding the human class I H chain HLA-B27 and human beta 2-microglobulin (beta 2m). In both cell types, we observed a large decrease in steady state levels specific for human class I H chains--both free H chains and those complexed with beta 2m. In the mouse cells transfected with HLA class I, infection did not affect levels and assembly of mouse class I H chains with human beta 2m. The effect of HCMV infection on class I expression is insensitive to phosphonoacetic acid, suggesting the involvement of immediate early or early viral proteins. Pulse-chase analysis showed that the low steady state level of class I H chains in HCMV-infected cells is not the result of a reduced rate of synthesis. Rather, we observed accelerated degradation of class I H chains, regardless of their association with beta 2m. We conclude that HCMV reduces human MHC class I protein levels by interference with the stability of class I H chains.
Tyrosine kinases of the Src family are synthesized as cytosolic proteins that subsequently translocate to membranes. Little is known of the mechanisms responsible for targeting these proteins to ...membranes, although a role for the cytosolic chaperone Hsp90 has been proposed. Here, we have studied the involvement of Hsp90 in the synthesis, membrane binding, and maintenance of the Src-kinase Lck. Using specific inhibitors of Hsp90, geldanamycin and radicicol, we found that functional Hsp90 is essential for the stability of newly synthesized, but not mature, Lck. Similar results were obtained for two other Src-kinases, c-Src and Lyn. In contrast, LckY505F and LckDeltaSH2, constitutively active Lck mutants lacking the C-terminal regulatory tyrosine or the entire Src homology 2 domain, respectively, required Hsp90 activity to stabilize the mature proteins. Lck synthesized in the absence of Hsp90 activity was degraded within 30-45 min. This unstable Lck was myristoylated normally but did not associate with membranes or CD4, interactions that normally start within minutes of the completion of Lck synthesis. A construct composed of the N-terminal unique domain of Lck fused to green fluorescent protein did not require Hsp90 activity during synthesis. In addition, this protein associated with membranes efficiently in the absence of Hsp90 activity. Together these data suggest that interaction with Hsp90 is necessary for the correct synthesis and subsequent membrane binding of Lck. However, Hsp90 does not appear to play a direct role in Lck membrane, or CD4, association.
MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine‐insensitive manner, suggesting that ...peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA‐DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation‐specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS‐stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post‐translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER‐derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.
Jan Hontelez and colleagues argue that the cost-effectiveness studies of HIV treatment scale-up need to include health system constraints to be more informative.
The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 ...and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.
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