System L-type transport of large neutral amino acids is mediated by ubiquitous LAT1-4F2hc and epithelial LAT2-4F2hc. These heterodimers are thought to function as obligatory exchangers, but only in¯ux properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, af®nity and exchange stoichiometry using the Xenopus oocyte expression system. Quanti®cation of amino acid in¯ux and ef¯ux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans-stimulations of amino acid in¯ux by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the ef¯ux selectivity range resembles that of in¯ux. Compared with high extracellular apparent af®nities, LAT1-and LAT2-4F2hc displayed much lower intracellular apparent af®nities (apparent K m in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1-4F2hc) and transcellular transport (LAT2-4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate af®nities such that the intracellular amino acid concentration controls their activity. Keywords: epithelial cell polarity/glycoproteinassociated amino acid transporter/LAT1-4F2hc/ LAT2-4F2hc IntroductionTwo heterodimeric transporters for large neutral amino acids that correspond to the Na + -independent system L have recently been identi®ed (Kanai et al., 1998;Mastroberardino et al., 1998;Pineda et al., 1999;Prasad et al., 1999;Rossier et al., 1999;Segawa et al., 1999;Rajan et al., 2000). These heterodimers contain catalytic subunits named LAT1 and LAT2, which belong to the family of glycoprotein-associated amino acid transporters (gpaATs) and are also called light chains (Verrey et al., , 2000. A disul®de bond covalently links these gpaATs to their associated glycoprotein 4F2hc/CD98.Functional experiments performed in expression systems suggest that the two L-type transporters function as exchangers (Mastroberardino et al., 1998;Pineda et al., 1999;Rossier et al., 1999). In the case of LAT2-4F2hc, some contradictory data have been published. Our laboratory and that of Palacin have shown that the ef¯ux of L-Phe and of L-Ile depend on the presence of an extracellular uptake substrate, as expected for an obligatory exchange (Pineda et al., 1999;Rossier et al., 1999). In contrast, an ef¯ux of L-Leu observed by Kanai and co-workers was interpreted as a facilitated diffusion (Segawa et al., 1999). Some functional differences between the two L-type transporters were reported, in particular the fact that LAT2-4F2hc has a broader selectivity range than LAT1-4F2hc in that it also mediates the uptake of smaller neutral amino acids.The tissue distribution and subcellular localization of the two L-type transporters suggest that they must play different roles. LAT1-4F2hc is found quite ubiquitously and is highly expressed in proliferating tissues, in particular also in tumors, sugges...

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Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.

Mating elicits two well-defined reactions in sexually matured females of many insects: reduction of receptivity and increased oviposition. These post-mating responses have been shown to be induced by factors synthesized in the reproductive tract of the adult male and transferred in the seminal fluid to the female during copulation. One of these factors, named sex-peptide (SP), has been identified in Drosophila melanogaster. Using an in vitro radiochemical assay, we show that synthetic sex-peptide considerably activates juvenile hormone III-bisepoxide (JHB3) synthesis in corpus allatum (CA) excised from Days 3 and 4 post-eclosion virgin females. Base levels are significantly lower at emergence (Day 0) than on subsequent days, and only weak stimulation is obtained on Day 1, while none is obtained on Day 2, where maximal basal synthesis occurs. The CA of mated females cannot be stimulated further for at least 7 days, but regain responsiveness by Day 10 after mating. Synthesis of JHB3 stimulated by SP in vitro persists for at least 4 h after removal of the peptide. Development of responsiveness of the CA to SP in vitro is compared with development of the post-mating reactions of sex-peptide injected virgin females. Our results suggest that the CA is a direct target for SP in vivo and that sexual maturity is established separately for the two post-mating reactions.