Early Embryonic Death of Mice Deficient in γ-Adaptin

Abstract

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the ␥-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. ␥-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free ␤1-or 1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.At several sites along the secretory and the endocytic routes the transport of membrane proteins and luminal cargo depends on the formation of carrier vesicles. The formation of these transport vesicles is facilitated by coat proteins. Budding from the Golgi and endoplasmic reticulum membranes involves the heptameric COP-I and COP-II complexes. Budding from the trans-Golgi network (TGN), 1 the plasma membrane, and also probably from membranes of the endosomal/lysosomal system involves heterotetrameric adaptor protein (AP) complexes of which three types are known at present and which show homology to the COP-I subunits (1-3).AP-1 and AP-2 have been implicated in the formation of clathrin-coated vesicles at the TGN and the plasma membrane, respectively, but may participate also in vesicle formation at other sites (3-5). AP-3-mediated vesicle formation may take place at the TGN and/or endosomes (6 -8).Although most of our knowledge on AP function is derived from studies on their subcellular localization and interaction with membranes or cytoplasmic domains of membrane proteins, little is based on in vivo functional studies or genetic approaches. Deletion of genes of AP subunits in yeast did not reveal the function of AP-1 and AP-2 but was informative in the case of AP-3. Mutations of the four AP-1 and AP-2 subunits did not result in a mutant phenotype and only the combination of AP-1 mutations with a temperature-sensitive clathrin heavychain mutation enhanced the reduced growth rate observed in the clathrin heavy-chain mutant (9 -11). 2 No biochemical data for cargo-AP-1 interactions are available in the yeast system. The AP-3 is needed for an alternative pathway from the TGN to the vacuole, which is utilized by the vacuolar alkaline phosphatase and the vacuolar t-SNARE Vam3p and does not depend on clathrin (12). In Drosophila melanogaster AP-3 is involved in the formation of pigment granules, as indicated by garnet, an eye color mutant caused by mutations in one of the AP-3 subunits (8, 13). Further support for a role of AP-...

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