Upon agonist stimulation, many G protein-coupled receptors such as ␤ 2 -adrenergic receptors are internalized via ␤-arrestin-and clathrin-dependent mechanisms, whereas others, like M 2 muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin-and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M 2 mAChR endocytosis, we investigated the post-endocytic trafficking of M 2 mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M 2 mAChR internalization. Here, we report that M 2 mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, G␣ q Q209L. In contrast, M 2 mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrindependent endocytosis. Nevertheless, M 2 mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M 2 mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.

Three bacterial isolates, Pseudomonas fluorescens F1, Pseudomonas rhodesiae R1 and Pseudomonas veronii V1 were genetically modified by introduction of a plasmid, pJH123, with a phoA hybrid gene that directed constitutive overproduction of the enzyme alkaline phosphatase. The presence of the plasmid in the bacterial hosts elevated extracytoplasmic alkaline phosphatase production from 100- to 820-fold. The growth and survival of the plasmid-bearing hosts in sterilized soil slurries was comparable to parental control strains. In the absence of antibiotic selection, pJH123 was maintained in two of the three hosts (P. fluorescens F1 and P. veronii V1) during incubation in minimal medium. The effects of the genetically enhanced pseudomonads on the liberation of inorganic phosphate (PO(4) (3-)) were determined in sterilized soil slurries following the addition of an organophosphorus compound, glycerol-3-phosphate. A significant accumulation of PO(4) (3-) was measured in soil slurries amended with 10 mM glycerol-3-phosphate and any of the three phosphatase-enhanced pseudomonad isolates. In contrast, soil slurries containing unmodified parental strains did not exhibit significant PO(4) (3-) accumulation. Two of the three enhanced phosphate-liberating strains released sufficient PO(4) (3-) that cell-free supernatants from sterilized soil slurry incubations removed significant amounts of uranium (as much as 69%) from solution.