Search

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.

The major coat proteins of clathrin-coated vesicles are the clathrin triskelion and heterotetrameric associated protein (AP) complexes. The APs are thought to be involved in cargo capture and recruitment of clathrin to the membrane during endocytosis and sorting in the trans-Golgi network/endosomal system. AP180 is an abundant coat protein in brain clathrin-coated vesicles, and it has potent clathrin assembly activity. In Saccharomyces cerevisiae, there are 13 genes encoding homologs of heterotetrameric AP subunits and two genes encoding AP180-related proteins. To test the model that clathrin function is dependent on the heterotetrameric APs and/or AP180 homologs, yeast strains containing multiple disruptions in AP subunit genes, as well as in the two YAP180 genes, were constructed. Surprisingly, the AP deletion strains did not display the phenotypes associated with clathrin deficiency, including slowed growth and endocytosis, defective late Golgi protein retention and impaired cytosol to vacuole/autophagy function. Clathrin-coated vesicles isolated from multiple AP deletion mutants were morphologically indistinguishable from those from wild-type cells. These results indicate that clathrin function and recruitment onto membranes are not dependent upon heterotetrameric adaptors or AP180 homologs in yeast. Therefore, alternative mechanisms for clathrin assembly and coated vesicle formation, as well as the role of AP complexes and AP180-related proteins in these processes, must be considered.

Among the various coats involved in vesicular transport, the clathrin associated coats that contain the adaptor complexes AP-1 and AP-2 are the most extensively characterized. The function of the recently described adaptor complex AP-3, which is similar to AP-1 and AP-2 in protein composition but does not associate with clathrin, is not known. By monitoring surface plasmon resonance we observed that AP-3 is able to interact with the tail of the lysosomal integral membrane protein LIMP-II and that this binding depends on a DEXXXLI sequence in the LIMP-II tail. Furthermore, AP-3 bound to the cytoplasmic tail of the melanosome-associated protein tyrosinase which contains a related EEXXXLL sequence. The tails of LIMP-II and tyrosinase either did not interact, or only interacted poorly, with AP-1 or AP-2. In contrast, the cytoplasmic tails of other membrane proteins containing di-leucine and/or tyrosine-based sorting signals did not bind AP-3, but AP-1 and/or AP-2. This points to a function of AP-3 in intracellular sorting to lysosomes and melanosomes of a subset of cargo proteins via di-leucine-based sorting motifs.

In Saccharomyces cerevisiae, the redundant YCK1 and YCK2 genes (Yeast Casein Kinase 1) are required for viability. We describe here the molecular analysis of four mutations that eliminate the requirement for Yck activity. These mutations alter proteins that resemble the four subunits of clathrin adaptors (APs), with highest sequence similarity to those of the recently identified AP-3 complex. The four yeast subunits are associated in a high-molecular-weight complex. These proteins have no essential function and are not redundant for function with other yeast AP-related proteins. Combination of suppressor mutations with a clathrin heavy chain mutation (chc1-ts) confers no synthetic growth defects. However, a yck(ts) mutation shows a strong synthetic growth defect with chc1-ts. Moreover, endocytosis of Ste3p is dramatically decreased in yck(ts) cells and is partially restored by the AP suppressor mutations. These results suggest that vesicle trafficking at the plasma membrane requires the activity of Yck protein kinases, and that the new AP-related complex may participate in this process.

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.

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 delta-adaptin, a structural homolog of the alpha- and gamma-adaptin subunits of the clathrin coat adaptors AP-1 and AP-2, respectively. Biochemical analyses demonstrated that delta-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 delta-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.

We have identified two closely related human proteins (sigma3A and sigma3B) that are homologous to the small chains, sigma1 and sigma2, of clathrin-associated adaptor complexes. Northern and Western blot analyses demonstrate that the products of both the sigma3A and sigma3B genes are expressed in a wide variety of tissues and cell lines. sigma3A and sigma3B 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 beta-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 sigma3-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 sigma3 chains are components of a novel, ubiquitous adaptor-like complex involved in the recognition of tyrosine-based sorting signals.

We have established by peptide mapping and immunochemical analysis of purified clathrin assembly protein preparations from bovine brain, that the cluster of components of mol. wt 100-120 kd fall into four classes, which we term alpha, beta, beta' and gamma. The beta and beta' proteins are immunologically related and generate a series of common tryptic peptides. The same criteria reveal no such homologies between the alpha, beta(beta') and gamma polypeptides. The so-called HA-II assembly protein group contains equimolar amounts of alpha and beta class polypeptides, which are shown to interact with each other. In the HA-I group assembly protein complex gamma and beta' class polypeptides form a stoichiometric complex. Immunofluorescence microscopy reveals that the HA-I complex is specifically associated with clathrin-coated membranes in the Golgi region of cultured cells, whereas the HA-II complex appears to be restricted to coated pits on the plasma membrane. The data lead to the tentative conclusion that the clathrin assembly proteins are involved in the recognition of the intracellular targets by uncoated vesicles.

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.