Abstract. We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide , as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30°C, FM 4-64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0°C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25°C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20--40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4-64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15°C delayed vacuolar labeling and trapped FM 4-64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4-64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in secl mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosomelike intermediate compartment (labeled at 15°C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro. ,•ECRETION and endocytosis are major mechanisms of membrane flow to and from the plasma membrane (PM) ~ in eukaryotic ceils. Plasma membrane equilibrium is maintained by the addition of secretory vesicles after fusion and subtraction of endocytic vesicles after invagination. Thus, eukaryotic cells must balance secretory and endocytic traffic to maintain the appropriate protein and lipid content in the PM. Studies on the secretory and endocytic pathways have contributed greatly to our present understandAddress all correspondence to T. A. Vida at his present address: Department of Pharmacology, University of Texas-Houston, Health Science Center, Houston, TX 77225. Ph

We are studying intercompartmental protein transport to the yeast lysosome-like vacuole with a reconstitution assay using permeabilized spheroplasts that measures, in an ATP and cytosol dependent reaction, vacuolar delivery and proteolytic maturation of the Golgi-modified precursor forms of vacuolar hydrolases like carboxypeptidase Y (CPY). To identify the potential donor compartment in this assay, we used subcellular fractionation procedures that have uncovered a novel membrane-enclosed prevacuolar transport intermediate. Differential centrifugation was used to separate permeabilized spheroplasts into 15K and 150K g membrane pellets. Centrifugation of these pellets to equilibrium on sucrose density gradients separated vacuolar and Golgi complex marker enzymes into light and dense fractions, respectively. When the Golgi-modified precursor form of CPY (p2CPY) was examined (after a 5-min pulse, 30-s chase), as much as 30-40% fractionated with an intermediate density between both the vacuole and the Golgi complex. Pulse-chase labeling and fractionation of membranes indicated that p2CPY in this gradient region had already passed through the Golgi complex, which kinetically ordered it between the Golgi and the vacuole. A mutant CPY protein that lacks a functional vacuolar sorting signal was detected in Golgi fractions but not in the intermediate compartment indicating that this corresponds to a post-sorting compartment. Based on the low transport efficiency of the mutant CPY protein in vitro (decreased by sevenfold), this intermediate organelle most likely represents the donor compartment in our reconstitution assay. This organelle is not likely to be a transport vesicle intermediate because EM analysis indicates enrichment of 250-400 nm compartments and internalization of surface-bound 35S-alpha-factor at 15 degrees C resulted in its apparent cofractionation with wild-type p2CPY, indicating an endosome-like compartment (Singer, B., and H. Reizman. 1990. J. Cell Biol. 110:1911-1922). Fractionation of p2CPY accumulated in the temperature sensitive vps15 mutant revealed that the vps15 transport block did not occur in the endosome-like compartment but rather in the late Golgi complex, presumably the site of CPY sorting. Therefore, as seen in mammalian cells, yeast CPY is sorted away from secretory proteins in the late Golgi and transits to the vacuole via a distinct endosome-like intermediate.

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 HermanskyPudlak 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 vacuole͞prevacuole-associated target soluble Nethylmaleimide-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.H ermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis in which oculocutaneous albinism, bleeding, and in most cases pulmonary fibrosis result from defects of melanosomes, platelet-dense granules, and lysosomes (1-4). Somewhat similar disorders, Chediak-Higashi and Griscelli syndromes, are additionally associated with severe immunodeficiency (2, 3). Important clues to the pathogenesis of these disorders have come from the mouse, in which Ͼ16 loci have been associated with mutant phenotypes similar to those of human HPS, Chediak-Higashi syndrome, and Griscelli syndrome (5, 6). Several of these genes have been identified recently and in a number of cases have been shown to result in homologous disorders in mice and humans (2-4). Although the functions of many of the corresponding gene products remain unknown, several are involved in various aspects of trafficking proteins to nascent organelles, particularly melanosomes, lysosomes, and cytoplasmic granules. In the yeast, Ͼ65 proteins have been implicated in biogenesis of the cytoplasmic vacuole, including the products of Ͼ40 vacuolar protein-sorting (vps) loci required for trafficking newly synthesized proteins from the late Golgi͞trans-Golgi network to the vacuole (7, 8). It seems likely that at least as many proteins are associated with organellar biogenesis in mammals.We have mapped and positionally cloned the mouse buff (bf ) locus, which is characterized by recessive coat-color hypopigmentation and mild platelet-storage pool deficiency but has little if any effect on lysosomal function. We find that mouse bf results from a missense substitution in Vps33a, a homologue of yeast vps33. The bf mutation results in defective melanosome morphology and melanogenesis both in vivo and in vitro. Expression of wild-type Vps33a in transfected mouse bf-mutant melanocytes complements this aberrant phenotype, whereas expression of bf-mutant Vps33a does not. These results establish murine bf a...

Abstract. Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud. The new bud vacuole is founded by a stream of vacuolederived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle. We now report in vitro formation of vacuole-derived tubules and vesicles. In semiintact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by FAC1 and VAC2, two genes which are required for vacuole inheritance in vivo. Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP. After forming tubulovesicular structures, isolated vacuoles subsequently increase in size. Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents. Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.