The signalling lipid PI(3,5)P2 is generated on endosomes and regulates retrograde traffic to the trans‐Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P2 levels. Mutations that lower PI(3,5)P2 cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P2 was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P2 regulatory complex by direct contact with the known regulators of PI(3,5)P2: Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P2 regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2. Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse. There is a [Have you seen ...?][1] (January 2009) associated with this Article. [1]: http://dx.doi.org/10.1038/emboj.2008.270

Considering the relatively small size of the HIV-1 genome, a sizable number of HIV-1 gene products have been implicated in interference with MHC class I Ag presentation. The strongest evidence is for roles for Nef and Tat in this process. Nef is a protein that is unnecessary for HIV-1 replication, but that is required for the development of the immune deficiencies associated with HIV infection (40). Nef increases the pathogenicity of HIV in several ways, including down-modulation of cell surface MHC class I molecules (28, 30). Specific regions of Nef that are involved in MHC class I down-regulation have been identified (29, 41). CTL killing of HIV-1-infected primary cells is inefficient if Nef is expressed, and the resistance of the infected cells is due to MHC class I down-regulation (42).

Tapasin influences the quantity and quality of MHC/peptide complexes at the cell surface; however, little is understood about the structural features that underlie its effects. Because tapasin, MHC class I, and TAP are transmembrane proteins, the tapasin transmembrane/cytoplasmic region has the potential to affect interactions at the endoplasmic reticulum membrane. In this study, we have assessed the influence of a conserved lysine at position 408, which lies in the tapasin transmembrane/cytoplasmic domain. We found that substitutions at position K408 in tapasin affected the expression of MHC class I molecules at the cell surface, and down-regulated tapasin stabilization of TAP. In addition to affecting TAP interaction with tapasin, the substitution of alanine, but not tryptophan, for the lysine at tapasin position 408 increased the amount of tapasin found in association with the open, peptide-free form of the HLA-B8 H chain. Tapasin K408A was also associated with more folded, β₂-microglobulin-assembled HLA-B8 molecules than wild-type tapasin. Consistent with our observation of a large pool of tapasin K408A-associated HLA-B8 molecules, the rate at which HLA-B8 migrated from the endoplasmic reticulum was slower in tapasin K408A-expressing cells than in wild-type tapasin-expressing cells. Thus, the alanine substitution at position 408 in tapasin may interfere with the stable acquisition by MHC class I molecules of peptides that are sufficiently optimal to allow MHC class I release from tapasin.

Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L^d/tapasin association can be segregated from reconstitution of folded L^d surface expression. This finding suggests that peptide acquisition by L^d is influenced by tapasin functions that are independent of L^d binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L^d, and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L^d. These data are consistent with a higher overall affinity between L^d and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin’s stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin’s electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.

Nucleotide excision repair is a major pathway for repairing UV light-induced DNA damage in most organisms. Using insertional mutagenesis, we isolated a UV-sensitive mutant of Chlamydomonas reinhardtii that is blocked in the excision of cyclobutane pyrimidine dimers. The mutant is also sensitive to the alkylating agent, methyl methanesulphonate. We have cloned REX1, a novel gene that rescues the mutant. The gene is unusual in a eukaryotic organism in that it is predicted to encode two different proteins, a small protein (8.9 kDa) and a larger protein (31.8 kDa). Neither protein is homologous to known DNA repair proteins. Partial complementation is achieved with subclones of the gene encoding only the 8.9-kDa protein. The 8.9-kDa protein has homologues in many organisms including Saccharomyces cerevisiae, Arabidopsis, and humans. The 31.8-kDa protein appears to be less conserved. These findings may be of general importance for DNA repair in other organisms.