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The provision of a good light source is important for reptiles. For instance, ultraviolet light is used in social interactions and used for vitamin D synthesis. With respect to housing, most reptilians are best kept pairwise or individually. Environmental enrichment can be effective but depends on the form and the species to which it is applied. Temperature gradients around preferred body temperatures allow accurate thermoregulation, which is essential for reptiles. Natural distributions indicate suitable ambient temperatures, but microclimatic conditions are at least as important. Because the nutrient requirements of reptiles are largely unknown, facilitating self-selection from various dietary items is preferable.

The retromer complex is involved in the retrograde transport of the CI-M6PR (cation-independent mannose 6-phosphate receptor) from endosomes to the Golgi. It is a hetero-trimeric complex composed of Vps26 (vacuolar sorting protein 26), Vps29 and Vps35 proteins, which are conserved in eukaryote evolution. Recently, elucidation of the crystal structure of Vps29 revealed that Vps29 contains a metallo-phosphoesterase fold [Wang, Guo, Liang, Fan, Zhu, Zang, Zhu, Li, Teng, Niu et al. (2005) J. Biol. Chem. 280, 22962–22967; Collins, Skinner, Watson, Seaman and Owen (2005) Nat. Struct. Mol. Biol. 12, 594–602]. We demonstrate that recombinant hVps29 (human Vps29) displays in vitro phosphatase activity towards a serine-phosphorylated peptide, containing the acidic-cluster dileucine motif of the cytoplasmatic tail of the CI-M6PR. Efficient dephosphorylation required the additional presence of recombinant hVps26 and hVps35 proteins, which interact with hVps29. Phosphatase activity of hVps29 was greatly decreased by alanine substitutions of active-site residues that are predicted to co-ordinate metal ions. Using inductively coupled plasma MS, we demonstrate that recombinant hVps29 binds zinc. Moreover, hVps29-dependent phosphatase activity is greatly reduced by non-specific and zinc-specific metal ion chelators, which can be completely restored by addition of excess ZnCl2. The binuclear Zn2+ centre and phosphate group were modelled into the hVps29 catalytic site and pKa calculations provided further insight into the molecular mechanisms of Vps29 phosphatase activity. We conclude that the retromer complex displays Vps29-dependent in vitro phosphatase activity towards a serinephosphorylated acidic-cluster dileucine motif that is involved in endosomal trafficking of the CI-M6PR. The potential significance of these findings with respect to regulation of transport of cycling trans-Golgi network proteins is discussed.

ABSTRACTT-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRα molecules, having a half-life of approximately 20 min in immature CD4+CD8+thymocytes compared with >75 min in mature T cells. The molecular basis for TCRα instability in CD4+CD8+thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRα proteins in all other T cell types examined. Here, we compared the processing of TCRα glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+thymocytes and splenic T cells. These studies show that TCRα glycoproteins synthesized in CD4+CD8+thymocytes were processed in a similar manner as those made in splenic T cells and that TCRα proteins stably associated with calnexin in both cell types. Interestingly, however, TCRα association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+thymocytes compared with splenic T cells. Finally, TCRα degradation in CD4+CD8+thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin·TCRα complexes. These data demonstrate that calnexin association is not sufficient to protect TCRα proteins from rapid degradation in CD4+CD8+thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRα glycoproteins.

Most T lymphocytes express on their surfaces an oligomeric protein complex consisting of clonotypic αβ polypeptides associated with invariant CD3-γδε and ζ chains, designated the T cell antigen receptor (TCR) complex. Assembly and intracellular transport of nascent TCR proteins is believed to be assisted by their interaction with the molecular chaperone calnexin, which for certain molecules functions as a lectin for monoglucosylated glycans. However, as most of our knowledge about calnexin-TCR protein associations has been obtained under conditions of limited TCR assembly, the role of calnexin in the formation of nascent TCR complexes is unclear. Here, we studied the role of glucose (Glc) trimming and calnexin association in the oligomerization of TCRα and CD3δ glycoproteins in murine splenic T lymphocytes, a model cell type for efficient assembly of complete TCR complexes. We show that removal of Glc residues from both CD3δ proteins and TCRα proteins occurred prior to their association with any other TCR components and that calnexin specifically interacted with unassembled TCRα and CD3δ proteins containing incompletely trimmed oligosaccharides. Interestingly, we found that removal of Glc residues from glycan chains was necessary for efficient association of calnexin with TCRα glycoproteins but not with CD3δ glycoproteins. These studies define Glc trimming and calnexin association as initial molecular events in the translation of CD3δ and TCRα proteins, occurring coincident with or immediately after their translocation into the endoplasmic reticulum and preceding the ordered pairing of TCR chains. In addition, these data document that calnexin assembly with CD3δ and TCRα glycoproteins involves both glycan-dependent and glycan-independent mechanisms.