A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.

Indolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (K K P L L P ). The genes iorA and iorB which encode respective K K and L L subunits, were coexpressed heterologously in Escherichia coli cells under anaerobic conditions. IOR activity was detected from the cell extract containing both subunits and its activity was enhanced by in vitro heat treatment prior to the assay. The iorA and iorB were expressed individually and each subunit was examined for enzymatic activity with and without heat treatment. IOR activity was detected neither from the extract of K K subunit nor L L subunit. The K K and L L subunits were mixed and then IOR activity was examined. Weak IOR activity was detected without heat treatment, however, upon heat treatment its activity was enhanced. The mixture of individually heat treated K K and L L subunits did not possess any IOR activity even though the mixed sample was heat treated again. IOR K K and L L subunits were individually purified to homogeneity, mixed with or without heat treatment and subunit assembly was examined by determining molecular mass. Upon heat treatment, inactive K K and L L were converted to an active high molecular weight complex (195 kDa) which corresponds to the K K P L L P structure. However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the heterooligomerization of IOR subunits.z 1998 Federation of European Biochemical Societies.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis. The transferrin receptor (TR)1 binds the serum iron transport protein transferrin (Tf), internalizes through clathrincoated pits, and facilitates Tf iron release in the sorting endosome. Efficient TR internalization requires a cytoplasmic tail tyrosine-containing motif, Tyr-Thr-Arg-Phe (1, 2). Studies by Ohno et al. (3) using yeast two-hybrid analysis demonstrate that the TR cytoplasmic tail signal interacts with one of the four subunits of the AP-2 adaptor complex, the 2 chain. This interaction provides a mechanism for promoting TR clustering in clathrin-coated pits and subsequent internalization. The AP-2 adaptor complex is also required for clathrin recruitment (4 -6) and lattice assembly (7) and, together with its direct interaction with receptor cytoplasmic tails, links the cell surface receptors to the clathrin-based endocytic machinery (reviewed in Refs. 8 and 9).Despite the extensive characterization of many of the proteins involved in endocytosis, little is known about how the clathrin-based endocytic machinery is regulated. What is evident, however, is endocytosis via clathrin-coated pits is blocked during mitosis (10), starting at prophase and continuing through telophase (11,12). In A431 cells, the mitotic block appears to arrest clathrin assembly at various stages of invagination (13), suggesting that the continuous activity of an enzyme (or enzymes) is required for vesicle formation (reviewed in Ref. 14). Using in vitro reconstitution assays, mitotic cytosol has been shown to inhibit invagination of clathrin-coated pits and one of the factors responsible is cdc2 kinase (15).The role of kinases in receptor trafficking has been demonstrated in studies on the asia...

The sorting of membrane proteins to the lysosome requires tyrosine-or dileucine-based targeting signals. Recycling receptors have similar signals, yet these proteins seldom enter the latter stages of the endocytic pathway. To determine how lysosomal and internalization signals differ, we prepared chimeric molecules consisting of the cytoplasmic tails of CD3 ␥-chain, lysosomal acid phosphatase, and lysosomal-associated membrane glycoprotein-1, each fused to the transmembrane and extracellular domains of the transferrin receptor (TR). Each chimera was expressed on the cell surface and rapidly internalized. Metabolic pulse-chase experiments showed that the CD3 ␥-chain and lysosomal acid phosphatase chimeras, unlike the lysosomal-associated membrane glycoprotein chimera, were rapidly de-

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