Defective expression of the μ3 subunit of the AP-3 adaptor complex in the Drosophila pigmentation mutant carmine

The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (μ) chain families μ1, μ2, and μ3, of the corresponding AP complexes, and δ-COP, the analogous component of the coatomer (COPI) complex. The Drosophila orthologs exhibit a high degree of sequence identity to mammalian medium chain and δ-COP proteins. Northern analysis demonstrated that medium chain and δ-COP mRNAs are expressed uniformly throughout fly development. Medium chain and δ-COP genes were cytologically mapped and the μ3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm ¹ mutant allele indicated the presence of a large insertion in the coding region of the μ3 gene and Northern analysis revealed no detectable μ3 mRNA. Light microscopy of the cm ¹ mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence of a role for μ3 in the sorting processes required for pigment granule biogenesis in Drosophila.     

The T cell receptor-associated protein is proteolytically cleaved in a pre-Golgi compartment

The TCR consists of at least seven transmembrane chains: the clonotypic disulfide-linked alpha- and beta-chains, the invariant gamma-, delta-, and epsilon-chains, termed the CD3 complex, and the zeta-zeta homodimer. We have recently described an additional 26-kDa protein, which is transiently associated with newly synthesized mouse CD3 chains in the endoplasmic reticulum. The exact function of this protein, which we called TRAP (for TCR-associated protein) is not yet known; studies suggest however, that it may play a role in the assembly of the TCR complex. Here we report the properties of another protein which has a Mr of 16,000 and, like TRAP, can be coimmunoprecipitated from metabolically labelled murine T cells with antibodies against the chains of the CD3 complex. Kinetic analysis suggests a precursor-product relationship between TRAP and the 16-kDa protein: the latter starts appearing once TRAP begin to disappear. Having reached a maximal level at approximately 1 h after biosynthesis, it is rapidly lost. Agents that slow or block the disappearance of TRAP, delay or prevent the appearance and eventual disappearance of the 16-kDa protein. Incubation of immunoprecipitates containing gamma, epsilon, and TRAP in vitro at 37 degrees C results in the appearance of the 16-kDa protein. Employing HPLC peptide mapping we demonstrate that this 16-kDa protein is structurally related to TRAP. These results suggest that the removal of TRAP from the newly synthesized CD3 chains is accompanied by its proteolytic cleavage in a pre-Golgi compartment.