Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.     

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.     

Shape transitions and shape stability of giant phospholipid vesicles in pure water induced by area-to-volume changes.

Shape transformations of vesicles of dimyristoylphosphatidylcholine (= DMPC) and palmitoyloleylphosphatidylcholine (= POPC) in ion-free water were induced by changing the area-to-volume ratio via temperature variations. Depending on the pretreatment we find several types of shape changes for DMPC (in pure water) at increasing area-to-volume ratio: (a) budding transitions leading to the formation of a chain of vesicles at further increase of the area-to-volume ratio, (b) discocyte-stomatocyte transitions, (c) reentrant dumbbell-pear-dumbbell transitions, and (d) spontaneous blebbing and/or tether formation of spherical vesicles. Beside these transitions a more exotic dumbbell-discocyte transition (e) was found which proceeded via local instabilities. Pears, discocytes, and stomatocytes are stable with respect to small temperature variations unless the excess area is close to values corresponding to limiting shapes of budded vesicles where temperature variations of less than or equal to 0.1 degree C lead to spontaneous budding to the inside or the outside. For POPC we observed only budding transitions to the inside leading either to chains of vesicles or to distributions of equally sized daughter vesicles protruding to the inside of the vesicle. Preliminary experiments concerning the effect of solutes are also reported. The first three types of shape transitions can be explained in terms of the bilayer coupling model assuming small differences in thermal expansivities of the two monolayers. This does not hold for the observed instabilities close to the limiting shapes.     

Taurine release associated to volume regulation in rabbit lymphocytes.

Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4 degrees C, but was unchanged at 15 degrees C or 25 degrees C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested.     

Benthic-pelagic flux rates on mussel beds: tunnel and tidal flume methodology compared

Material flux rates in an intertidal mussel bed were measured synchronously over two tidal cycles in June 1989 with Benthic Ecosystem Tunnels and a double lane flume. The tunnels enclosed the near bottom water, whereas the flume canalized the total water column. One tunnel was set up in a mussel bed and another one in an adjacent sand bottom as a control. The flume enclosed a mussel lane and a sand lane. In the tunnel and in the flume the mussel bed revealed ammonium and phosphate discharge. At the same time, phytoplankton, dominated byPhyaeocystis globosa, was taken up intensively. These flux rates showed the same tendency but they were higher in the flume than in the tunnel. Different tendencies and flux rates for oxygen and particulate organic matter (POC, PN) were found in flume and tunnel. These differences demonstrate the importance of water column processes regarding the material exchange of a mussel bed. Tunnels enclose smaller bodies of water and are therefore expected to detect even small effects of the benthos on the passing water. In flumes, benthic influence may be diluted over the entire water column but conditions are more natural. The use of flumes is restricted to shallow waters while tunnels have the potential to be used at any depth.     

Total Thyroxine and Free Thyroxine Index in Plasma of Dairy Cows in Relation To Strength of Heat

Total thyroxine (TT4) and free thyroxine index (FT4I) were measured in peripheral plasma of cows. The samples were collected at the time of insemination from 66 cows showing pronounced signs of the heat and from 56 cows showing weak or silent heat. Neither TT4 or FT4I in plasma differed significantly between the two categories of oestrous cows.     

Differential mitotic rates and patterns of growth in compartments in the Drosophila wing

In the wing disks of Drosophila slowly dividing cells of Minute mutations are progressively eliminated from Minute/Minute⁺ mosaic compartments by a process known as cell competition. From a study of two different Minutes we show here that the intensity of competition is greater in the more extreme Minute with the slowest rate of cell division. The way in which the more rapidly growing Minute⁺ clones grow and overcome the surrounding Minute cells is described and cell competition is shown to be a result of local interactions between slow- and faster-growing cells.     

Structural studies of the apoprotein of the Folch-Pi bovine brain myelin proteolipid : characterization of the CNBr-fragments and of a long C-terminal sequence.

The action of cyanogen bromide on the quite insoluble bovine proteolipid apoprotein allowed the determination of four peptide fragments: two of them constituted a 19 amino acid long C-terminal sequence of the apoprotein. Our results were in favour of the existence of only one subunit presenting a molecular weight closely related to 25,000 for which a schematic representation is given.