Richard T. Swank

The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation.

In the mouse, more than 16 loci are associated with mutant phenotypes that include defective pigmentation, aberrant targeting of lysosomal enzymes, prolonged bleeding, and immunodeficiency, the result of defective biogenesis of cytoplasmic organelles: melanosomes, lysosomes, and various storage granules. Many of these mouse mutants are homologous to the human Hermansky–Pudlak syndrome (HPS), Chediak–Higashi syndrome, and Griscelli syndrome. We have mapped and positionally cloned one of these mouse loci, buff (bf), which has a mutant phenotype similar to that of human HPS. Mouse bf results from a mutation in Vps33a and thus is homologous to the yeast vacuolar protein-sorting mutant vps33 and Drosophila carnation (car). This is the first found defect of the class C vacuole/prevacuole-associated target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) complex in mammals and the first mammalian mutant found that is directly homologous to a vps mutation of yeast. VPS33A thus is a good candidate gene for a previously uncharacterized form of human HPS.

Cocoa: a new mouse model for platelet storage pool deficiency

We describe genetic, haematological and biochemical properties of a new mouse pigment mutant, cocoa (coa). Cocoa is a recessive mutation located on the centromeric end of chromosome 3 near the Car‐2 locus. The mutation causes increased bleeding time accompanied by symptoms of platelet storage pool deficiency (SPD), including decreased platelet serotonin and decreased visibility of dense granules as analysed by electron microscopy of unfixed platelets. Dense granules were visible in normal numbers when platelets were incubated with the fluorescent dye, mepacrine. The intragranular environment, however, was abnormal as indicated by decreased flashing of mepacrine‐loaded dense granules after exposure to ultraviolet light. Unlike the previously described seven mouse pigment mutations with SPD in which pigment granules, platelet dense granules and lysosomes are affected, the cocoa mutant had normal secretion of lysosomal enzymes from kidney proximal tubule cells and platelets. The cocoa mutation thus represents an example of a single gene which simultaneously affects melanosomes and platelet dense granules but probably does not affect lysosomes. The results indicate that melanosomes and platelet dense granules share steps in synthesis and/or processing. Cocoa may be a model for cases of human Hermansky‐Pudlak syndrome in which functions of melanosomes and platelet dense granules, but not lysosomes, are involved.

Involvement of the esterase active site of egasyn in compartmentalization of β-glucuronidase within the endoplasmic reticulum

Organophosphorous compounds, which are potent inhibitors of egasyn-esterase activity, caused a rapid dissociation of the high molecular weight egasyn-microsomal beta-glucuronidase complex when administered in vivo or when added in vitro to microsomal suspensions. The dissociation was relatively specific to phosphodiester inhibitors of the esterase active site. Also, the egasyn-esterase active site was inaccessible to substrates and to inhibitors when egasyn was complexed to beta-glucuronidase. Dissociation of the egasyn-microsomal beta-glucuronidase complex in vivo by organophosphorous compounds was followed by massive and rapid secretion of microsomal beta-glucuronidase, but not egasyn, into plasma. These experiments implicate the egasyn-esterase active site in attachment of microsomal beta-glucuronidase to egasyn by a novel mechanism that, in turn, compartmentalizes beta-glucuronidase within the endoplasmic reticulum.

Identity of esterase-22 and egasyn, the protein which complexes with microsomal β-glucuronidase

Recent experiments have demonstrated that egasyn not only sequesters β-glucuronidase in microsomes by forming high molecular weight complexes with β-glucuronidase, but also has carboxyl esterase activity. We have found several new phenotypes of egasyn-esterase after electrophoresis and isoelectric focusing of liver homogenates and purified egasyn of inbred and wild mouse strains. Several phenotypes corresponded in relative mobility and relative isoelectric point among inbred strains to that recently reported for esterase-22 by Eisenhardt and von Deimling [(1982). Comp. Biochem. Physiol. 73B:719]. This genetic evidence, plus a wide variety of comparative biochemical and physiological data, indicates that egasyn is identical to esterase-22. Both parental types of egasyn isozymes are expressed in heterozygous F1 progeny, suggesting that alterations in the egasyn structural gene are responsible for the altered isoelectric points. Also, egasyn is a monomer since no new esterase bands appear in F1 progeny. The variants in isoelectric point of egasyn map at or near the egasyn (Eg) gene within the esterases of cluster 1 near Es-9 on chromosome 8.

Altered secretion of kidney lysosomal enzymes in the mouse pigment mutants ruby-eye, ruby-eye-2-J, and maroon.

Melanosomes and lysosomes share structural and biosynthetic properties. Three mouse pigment mutants, ruby-eye, ruby-eye-2-J, and maroon, have abnormally high concentrations of kidney lysosomal enzymes. Concentrations of kidney nonlysosomal enzymes and of liver and serum lysosomal enzymes are normal. By light microscopy the mutants have normal kidney lysosome morphology. It does not appear that the mutant genes cause an increased rate of production of lysosomes since the increased kidney β-glucuronidase concentration is not accompanied by a corresponding increase in rate of synthesis. The common defect in all mutants is a decreased rate of secretion of lysosomal enzymes from kidney into urine. Eight mouse pigment mutants are now known which affect both melanosome and lysosome function. They should serve as useful models for the study of the biogenesis, structure, and processing of these and other subcellular organelles.

The synthesis and processing of β-glucuronidase in normal and egasyn deficient mouse kidney

Abstract The presence of a precursor form of β-glucuronidase, with a subunit molecular weight of 75,000 was demonstrated in mouse kidney. This was later processed to the mature form, with subunit molecular weight of 71,500. Tissue fractionation revealed that the precursor was associated with the microsomes whereas the mature form was associated with the lysosomes. In mice lacking egasyn both forms of β-glucuronidase were present, but the rate of processing was elevated compared to normal.

Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid.

SummaryThe size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparison, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit decreased lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver β-glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of β-glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gusn. Liver β-glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of β-glucuronidase in Gusn/Gusn mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. β-Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal β-glucuronidase. The loss of liver lysosomal β-glucuronidase activity was shown by immunotitration to be due to a decrease in the number of β-glucuronidase molecules in lysosomes of the congenic strain. The Gusn structural alteration likely causes the lowered lysosomal β-glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gusn/Gusb animals had intermediate levels of liver β-glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gusn/Gusn mice. Gusn is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.

GENETICS OF LYSOSOMAL FUNCTIONS**Aided by a grant from the National Foundation-March of Dimes and by N.I.H. grant GM-19521 from the United States Public Health Service.

Publisher Summary This chapter describes the genetics of lysosomal functions. It is often assumed that hydrolases with acidic pH optima are localized in lysosomes. The α- D -mannosidases represent a group of enzymes that at first glance might have some bearing on the question of the biosynthesis of lysosomal enzymes. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three liver enzymes are immunologically unrelated. β-Glucuronidase is an unusual lysosomal enzyme in that it is also present in considerable amounts in the endoplasmic reticuiurn of liver and kidney. Glucose and galactose are also present in all of the enzymes, and fucose is present in all of the rat enzymes but not in mouse β-glucuronidase.