Chean Eng Ooi

AP-3: an adaptor-like protein complex with ubiquitous expression

We have identified two closely related human proteins (σ3A and σ3B) that are homologous to the small chains, σ1 and σ2, of clathrin‐associated adaptor complexes. Northern and Western blot analyses demonstrate that the products of both the σ3A and σ3B genes are expressed in a wide variety of tissues and cell lines. σ3A and σ3B are components of a large complex, named AP‐3, that also contains proteins of apparent molecular masses of 47, 140 and 160 kDa. In non‐neuronal cells, the 47 kDa protein most likely corresponds to the medium chain homolog p47A, and the 140 kDa protein is a homolog of the neuron‐specific protein β‐NAP. Like other members of the medium‐chain family, the p47A chain is capable of interacting with the tyrosine‐based sorting signal YQRL from TGN38. Immunofluorescence microscopy analyses show that the σ3‐containing complex is present both in the area of the TGN and in peripheral structures, some of which contain the transferrin receptor. These results suggest that the σ3 chains are components of a novel, ubiquitous adaptor‐like complex involved in the recognition of tyrosine‐based sorting signals.

Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis.

The cell surface protein repertoire needs to be regulated in response to changes in the extracellular environment. In this study, we investigate protein turnover of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p, in response to a change in extra‐cellular copper levels. As Ctr1p mediates high affinity uptake of copper into the cell, modulation of its expression is expected to be involved in copper homeostasis. We demonstrate that Ctr1p is a stable protein when cells are grown in low concentrations of copper, but that exposure of cells to high concentrations of copper (10 microM) triggers degradation of cell surface Ctr1p. This degradation appears to be specific for Ctr1p and does not occur with another yeast plasma membrane protein tested. Internalization of some Ctr1p can be seen when cells are exposed to copper. However, yeast mutant strains defective in endocytosis (end3, end4 and chc1‐ts) and vacuolar degradation (pep4) exhibit copper‐dependent Ctr1p degradation, indicating that internalization and delivery to the vacuole is not the principal mechanism responsible for degradation. In addition, a variant Ctr1p with a deletion in the cytosolic tail is not internalized upon exposure of cells to copper, but is nevertheless degraded. These observations indicate that proteolysis at the plasma membrane most likely explains copper‐dependent turnover of Ctr1p and point to the existence of a novel pathway in yeast for plasma membrane protein turnover.

Altered expression of a novel adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet.

Drosophila eye pigmentation defects have thus far been attributed to mutations in genes encoding enzymes required for biosynthesis of pigments and to ABC‐type membrane transporters for pigments or their precursors. We report here that a defect in a gene encoding a putative coat adaptor protein leads to the eye color defect of garnet mutants. We first identified a human cDNA encoding δ‐adaptin, a structural homolog of the α‐ and γ‐adaptin subunits of the clathrin coat adaptors AP‐1 and AP‐2, respectively. Biochemical analyses demonstrated that δ‐adaptin is a component of the adaptor‐like complex AP‐3 in human cells. We then isolated a full‐length cDNA encoding the Drosophila ortholog of δ‐adaptin and found that transcripts specified by this cDNA are altered in garnet mutant flies. Examination by light and electron microscopy indicated that these mutant flies have reduced numbers of eye pigment granules, which correlates with decreased levels of both pteridine (red) and ommachrome (brown) pigments. Thus, the eye pigmentation defect in the Drosophila garnet mutant may be attributed to compromised function of a coat protein involved in intracellular transport processes required for biogenesis or function of pigment granules.

β3A-adaptin, a Subunit of the Adaptor-like Complex AP-3

Recent studies have described a widely expressed adaptor-like complex, named AP-3, which is likely involved in protein sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of the subunits of this complex, which we call β3A-adaptin. The predicted amino acid sequence of β3A-adaptin reveals that the protein is closely related to the neuron-specific protein β-NAP (61% overall identity) and more distantly related to the β1- and β2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that β3A-adaptin has a domain organization similar to β-NAP and to β1- and β2-adaptins. β3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that β3A-adaptin corresponds to the ∼140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being δ-adaptin, p47A (now called μ3A) and ς3 (A or B). β3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. β3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of β3A-adaptin reported here lend support to the idea that AP-3 is a structural and functional homolog of the clathrin-associated adaptors AP-1 and AP-2.

Cloning, expression, and localization of a novel γ-adaptin-like molecule

We describe the cloning, expression, and localization of γ2‐adaptin, a novel isoform of γ‐adaptin. The predicted human and mouse γ2‐adaptin proteins are ∼90 kDa and 64.4% and 61.7% identical to γ‐adaptin, respectively. γ2‐Adaptin was localized to the Golgi, its localization distinct from γ‐adaptin. The membrane association of γ‐ and γ2‐adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, γ2‐adaptin binding being insensitive to drug treatment. Together, these results suggest that γ2‐adaptin plays a role in membrane transport distinct from that played by γ‐adaptin.