The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives β-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). β-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 μM, while the IC50 for Escherichia coli and human RNases H was 50 μM and 5.7 μM, respectively. In contrast, the related tropolone analog β-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 μM and 40 μM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both β-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 μM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between β-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

The medium () chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXØ) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit 4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using 4 as a selector protein. Statistical analyses of the results revealed that 4 prefers aspartic acid at position Y؉1, proline or arginine at Y؉2, and phenylalanine at Y؊1 and Y؉3 (Ø). In addition, we examined the interaction of 4 with naturally occurring YXXØ signals by both two-hybrid and in vitro binding analyses. These experiments showed that 4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the 4-YXXØ interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a 4-specific YXXØ signal. This chimera was targeted to the endosomallysosomal system without being internalized from the plasma membrane.The heterotetrameric adaptor protein (AP) 1 complexes AP-1, AP-2, AP-3, and AP-4 are components of protein coats that associate with the cytosolic face of organelles of the secretory and endocytic pathways (reviewed in Refs. 1-4). AP-2 is associated with the plasma membrane and mediates rapid internalization of endocytic receptors, whereas AP-1, AP-3, and AP-4 are associated with the trans-Golgi network and/or endosomes and mediate intracellular sorting events. AP complexes are thought to participate in protein sorting by inducing the formation of coated vesicles as well as concentration of cargo molecules within the vesicles. Concentration of integral membrane proteins is mediated by direct interaction of the AP complexes with sorting signals present within the cytosolic tails of the proteins. Several types of cytosolic sorting signals have been described, the most common of which are referred to as "tyrosine-based" or "dileucine-based" depending on which residues are critical for activity (5, 6).The four AP complexes have a similar structure and are composed of two large chains (␣/␥/␦/⑀ and ␤1-4, 90 -130 kDa), a medium chain (1-4, ϳ50 kDa), and a small chain (1-4, ϳ20 kDa), each of which subserves a different function. Extensive analyses of the ␣ chain of AP-2 have shown that it interacts, either directly or indirectly, with many regulators of coat assembly and/or vesicle formation (7). By analogy, the ␥/␦/⑀ chains are presumed to interact with other proteins that play similar regulatory roles. ␤1, ␤2, and ␤3 interact with the scaffolding protein, clathrin (8 -10). In addition, ␤1 and ␤2 have been found to bind a subset of dileucine-based sorting signals (11). The chains, on the other hand, function as recognition molecules for signals con...

The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (