The adhesion molecule L-selectin is cleaved rapidly from the surface of activated leukocytes by tumor necrosis factor-␣ converting enzyme, a cell surface metalloprotease, and also undergoes slower constitutive shedding in unactivated cells. The structural features that render it susceptible to shedding are poorly understood. We therefore analyzed the shedding of a series of mutant and chimeric L-selectin molecules. Although murine L-selectin is cleaved at a specific location in the juxtamembrane region 11 amino acids distal to the cell membrane, this cleavage has little sequence specificity. However, proline substitution at the P2 or P3 position or deletion of the epidermal growth factor (EGF) domain completely blocks the rapid phorbol ester-induced cleavage, but does not affect the slower basal proteolytic shedding. Insertion of the 15-residue membrane-proximal region (MPR) of L-selectin into the heterologous protein B7.2 results in a molecule that undergoes constitutive proteolytic turnover. In contrast, insertion of both the EGF domain and the MPR confers susceptibility to both slow constitutive shedding and the rapid proteolytic cleavage induced by phorbol 12-myristate 13-acetate. These results demonstrate that constitutive and induced L-selectin cleavage are separable processes and that the rapid phorbol ester-induced shedding requires the presence of the EGF domain, a sequence that is remote from the cleavage site.The extracellular domains of many integral membrane glycoproteins undergo proteolytic cleavage and release from the cell surface into the surrounding fluid phase. The biologically significant proteins released in this manner include a number of lymphokines, growth factors, transcription factors, and adhesion molecules (reviewed in Refs. 1-4). Proteolytic cleavage is responsible for the regulated secretion of several cytokines derived from transmembrane precursors (5-8), and the shedding of surface receptors results in the desensitization of responsiveness to various cytokines (9 -12). Membrane protein shedding is also implicated in several disease processes. Inherited mutations in the p55 TNF-␣ 1 receptor lessen its sensitivity to proteolysis, cause its decreased proteolytic clearance from the cell surface, and result in a family of inherited autoinflammatory syndromes (13). Cleavage of the amyloid precursor protein (APP) at the ␤ and ␥ sites, releasing the amyloidogenic A␤ fragment, is implicated in the pathogenesis of Alzheimer's disease, and mutations that increase secretion of this peptide are associated with familial Alzheimer's disease (14 -16). The enzymes responsible for such cleavage are collectively referred to as membrane secretases or sheddases. A common mechanism for the secretion of many of these proteins has been inferred from the ability to inhibit their release with new hydroxamic acid-derived metalloprotease inhibitors (17-21) and by the identification of a mutant cell line that is defective in cleaving multiple proteins (22). More recently, a plasma membrane enzyme responsible ...

HE Minutes comprise a series of dominant factors discovered by BRIDGES and T MORGAN in 1923. The phenotype of all heterozygous Minutes is characterized by short, slender bristles and a longer developmental period than the wild type. In some cases, there may be secondary effects such as smaller body size, somewhat rougher eyes, thinner wings with a tendency toward plexate venation and sterility or low fertility, particularly in the female. All Minutes are lethal in the homozygous condition (BRIDGES and BREHME 1944). In addition, certain Minutes have been found to increase the frequency of somatic segregation and crossing over (BRIDGES 192.5; AIORGAX, BRIDGES and STURTEVAKT 1925;STERN 1936).

No abstract

Total soluble protein present in 48-, 60-and 72-hour larval stages of Drosophik melanogaster was determined for controls and for heterozygotes of the following Minute mutants: M(2)1, M(2)173, M(2)z, M(2)S-10, M(3)w, M(3)124, M(3)B2, M(3)y and M ( 3 ) l . Cytochrome c oxidase activity was also assayed at these stages of development in mitochondria isolated from whole larvae of each genotype.The results indicate that all of these mutants possess a common retardation in the rate of growth throughout the larval period. The different Minutes, however, each had distinctive growth patterns, often characterized by particular lags occurring at specific stages. The concentration of cytochrome c oxidase was not necessarily lowered in conjunction with periods of especially retarded growth, but instead, activity higher than that of the control was frequently found. Only one mutant of those tested, M(2)z, showed a significantly lower concentration of this enzyme.

Uptake into blood and incorporation into tissue protein of cystine-3% were assayed in larvae of controls and of five heterozygous Minute mutants of Drosophila melanogaster. The mutants studied were M(2)S-10, M(2)1*, M(2)z, M(3)y and M(3)w. In all mutants, the level of cystine in the blood was significantly higher than in controls and differences in concentration between these mutants were evident. All mutants incorporated cystine into protein, although the specific activities obtained were dependent on genotype. The fraction of the cystine in the blood pool which was incorporated into protein was directly correlated with viability and rate of growth. In contrast, the radioactivity of leucine-l-14C, both in blood and in tissue proteins, was not different in M(2)12 and M(3)w heterozygotes as compared to controls, although in M(2)12 the fraction of leucine in the blood pool incorporated into protein was lowered. It was concluded that these mutants possessed a common inability to withdraw cystine (or other sulfur-containing compounds) from the blood into various metabolic pathways. Feeding activity, digestion and absorption of nutrients appeared normal in these mutants.