Loss of Vacuolar H+-ATPase Activity in Organelles Signals Ubiquitination and Endocytosis of the Yeast Plasma Membrane Proton pump Pma1p

Yeast mutants lacking the intracellular V-ATPase proton pump (vma mutants) have reduced levels of the Pma1p proton pump at the plasma membrane and increased levels in organelles including the vacuolar lumen. We examined the mechanism and physiological consequences of Pma1p mislocalization. Pma1p is ubiquitinated in vma mutants, and ubiquitination depends on the ubiquitin ligase Rsp5p and the arrestin-related adaptor protein Rim8p. vma mutant strains containing rsp5 or rim8 mutations maintain Pma1p at the plasma membrane, suggesting that ubiquitination is required for Pma1p internalization. Acute inhibition of V-ATPase activity with concanamycin A triggers Pma1p ubiquitination and internalization. In an endocytosis-deficient mutant (end4Δ) Pma1p is ubiquitinated but retained at the plasma membrane during concanamycin A treatment. Consistent with specificity in signaling loss of V-ATPase activity to Pma1p, another plasma membrane transporter, Mup1p, is not internalized in a vma mutant, and loss of the Mup1p adaptor Art1p does not prevent Pma1p internalization in a vma mutant. Very poor growth of vma2 rsp5-1 and vma2 rim8Δ double mutants suggests that Pma1p internalization benefits the vma mutants. We hypothesize that loss of V-ATPase-mediated organelle acidification signals ubiquitination, internalization, and degradation of a portion of Pma1p as a means of balancing overall pH homeostasis.     

Pma1, a P-type Proton ATPase,Is a Determinant of Chronological Life Span in Fission Yeast

Chronological life span is defined by how long a cell can survive in a non-dividing state. In yeast, it is measured by viability after entry into stationary phase. To date, some factors affecting chronological life span have been identified; however, the molecular details of how these factors regulate chronological life span have not yet been elucidated clearly. Because life span is a complicated phenomenon and is supposedly regulated by many factors, it is necessary to identify new factors affecting chronological life span to understand life span regulation. To this end, we have screened for long-lived mutants and identified Pma1, an essential P-type proton ATPase, as one of the determinants of chronological life span. We show that partial loss of Pma1 activity not only by mutations but also by treatment with the Pma1 inhibitory chemical vanadate resulted in the long-lived phenotype in Schizosaccharomyces pombe. These findings suggest a novel way to manipulate chronological life span by modulating Pma1 as a molecular target.     

The Exomer Coat Complex Transports Fus1p to the Plasma Membrane via a Novel Plasma Membrane Sorting Signal in Yeast

Sorting of transmembrane cargo proteins to different cellular compartments is mediated by sorting signals that are recognized by coat proteins involved in vesicle biogenesis. We have identified a sorting signal in the yeast cell fusion protein Fus1p that is required for its transport from the trans-Golgi compartment to the plasma membrane. Transport of Fus1p from the trans-Golgi to the cell surface is dependent on Chs5p, a component of the multisubunit exomer complex. We show that Fus1p transport is also dependent on the exomer components Bch1p and Bud7p. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Fus1p localization to the cell surface in the absence of exomer, possibly by promoting an alternate, exomer-independent route of transport. Mutation of an IXTPK sequence in the cytosolic tail of Fus1p abolishes its physical interaction with Chs5p, results in mislocalization of Fus1p, and therefore causes a cell fusion defect. These defects are suppressed by disruption of AP-1. We suggest that IXTPK comprises a novel sorting signal that is recognized and bound by exomer leading to the capture of Fus1p into coated vesicles en route to the cell surface.     

大豆下胚轴质膜H+-ATPase质子转运的测定

以大豆下胚轴为材料,采用改进的匀浆介质,通过两相法制得具有质子转运活力的高纯度质膜微囊.并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率.质子载体和质子转运特性分析表明,由Mg^2＋-ATP引发的荧光猝灭可以被质子载体CCCP恢复,并被质子通道抑制剂DCCD抑制;并且发现质膜H^＋-ATPase专一抑制剂钒酸钠可以完全抑制荧光猝灭,同时发现荧光猝灭依赖于Mg^2＋,并受K^＋刺激,最适pH为6.5.以上证明所测荧光猝灭是由质膜H^＋-ATPase所进行的质子转运引起的.结果同时表明,维持H^＋-ATPase合适构象和提高质膜微囊封闭性是制备具有H^＋转运活力质膜微囊的两个关键因素.     

Yeast Plasma Membrane Vesicles Suitable for Transport

Isolated yeast plasma membrane vesicles demonstrate a permeability barrier toward K(+) and glucose. Influx and efflux of glucose are inhibited by UO(2) (2+) ions.     

A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration

We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H⁺-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi.     

Proteolytic Function of GPI-Anchored Plasma Membrane Protease Yps1p in the Yeast Vacuole and Golgi

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.     

Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein,Requires Tandem C2 Domains for Delivery to the Plasma Membrane

The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C₂A and C₂B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C₂A-C₂B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C₂B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.     

Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins,Mmm1p and Mdm10p

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.Mitochondria are indispensable organelles for normal eukaryotic cell function. Since mitochondria cannot be synthesized de novo, these organelles are inherited, i.e., transferred from mother to daughter during cell division. In the yeast Saccharomyces cerevisiae, vegetative cell division occurs by budding, a form of proliferation in which growth is directed toward the developing bud. Previous studies indicate that mitochondria undergo a series of cell cycle–linked motility events during normal inheritance in yeast (Simon et al., 1997). These are: (a) polarization of mitochondria towards the site of bud emergence in G₁ phase; (b) linear, polarized movement of mitochondria from mother cells to developing buds in S phase; (c) immobilization of newly inherited mitochondria in the bud tip during S and G₂ phases; and (d) release of immobilized mitochondria from the bud tip during M phase.There is mounting evidence that the actin cytoskeleton controls mitochondrial morphology and inheritance during vegetative yeast cell growth. The two major actin structures of yeast observed by light microscopy are patches and cables. Actin cables are bundles of actin filaments that extend from the mother into the bud. Mitochondria colocalize with these actin cables (Drubin et al., 1993; Lazzarino et al., 1994). Moreover, mutations such as deletion of the tropomyosin I gene, TPM1, or the mitochondrial distribution and morphology gene, MDM20, which selectively destabilize actin cables, result in the loss of polarized mitochondrial movement and reduce transfer of mitochondria into buds (Herman et al., 1997; Simon et al., 1997). Together, these studies indicate that normal mitochondrial inheritance in yeast requires association of mitochondria with actin cables.Cell-free studies reveal a possible mechanism underlying actin control of mitochondrial inheritance. Sedimentation assays document binding of mitochondria to the lateral surface of F-actin. This mitochondrial actin-binding activity is ATP-sensitive, saturable, reversible, and mediated by protein(s) on the mitochondrial surface (Lazzarino et al., 1994). In addition, ATP-driven, actin-dependent motor activity has been identified on the surface of mitochondria (Simon et al., 1995). These observations support a model of mitochondrial inheritance whereby mitochondria use an actin-dependent motor to drive their movement from mother to daughter cells along actin cable tracks.Yeast genetic screens have revealed several genes, collectively referred to as mdm (mitochondrial distribution and morphology) and mmm (maintenance of mitochondrial morphology), which are required for mitochondrial inheritance (McConnell et al., 1990; Burgess et al., 1994; Sogo and Yaffe, 1994). We have focused on two of these genes: MDM10 and MMM1. Deletion of MDM10 leads to the development of giant spherical mitochondria, presumably by the collapse of elongated mitochondria into a spherical mass (Sogo and Yaffe, 1994). Deletion of MMM1 (Burgess et al., 1994) produces a similar phenotype. In both mutants, the fraction of buds without mitochondria is high, indicating defective mitochondrial inheritance. The proteins encoded by these genes, Mdm10p and Mmm1p, appear to be integral membrane proteins in the mitochondrial outer membrane. Here, we report tests of the hypothesis that Mmm1p and Mdm10p are required to link mitochondria to the cytoskeleton.     

Ssy1p and Ptr3p Are Plasma Membrane Components of a Yeast System That Senses Extracellular Amino Acids

Mutations in SSY1 and PTR3 were identified in a genetic selection for components required for the proper uptake and compartmentalization of histidine in Saccharomyces cerevisiae. Ssy1p is a unique member of the amino acid permease gene family, and Ptr3p is predicted to be a hydrophilic protein that lacks known functional homologs. Both Ssy1p and Ptr3p have previously been implicated in relaying signals regarding the presence of extracellular amino acids. We have found that ssy1 and ptr3 mutants belong to the same epistasis group; single and ssy1 ptr3 double-mutant strains exhibit indistinguishable phenotypes. Mutations in these genes cause the nitrogen-regulated general amino acid permease gene (GAP1) to be abnormally expressed and block the nonspecific induction of arginase (CAR1) and the peptide transporter (PTR2). ssy1 and ptr3 mutations manifest identical differential effects on the functional expression of multiple specific amino acid transporters. ssy1 and ptr3 mutants have increased vacuolar pools of histidine and arginine and exhibit altered cell growth morphologies accompanied by exaggerated invasive growth. Subcellular fractionation experiments reveal that both Ssy1p and Ptr3p are localized to the plasma membrane (PM). Ssy1p requires the endoplasmic reticulum protein Shr3p, the amino acid permease-specific packaging chaperonin, to reach the PM, whereas Ptr3p does not. These findings suggest that Ssy1p and Ptr3p function in the PM as components of a sensor of extracellular amino acids.     

Plasma Membrane Localization of Gαz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, α_z. Wild-type α_z (α_z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does α_z-WT. Removal of the myristoylation site produced a mutant α_z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on α_z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on α_z-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on α_z specific localization at the plasma membrane.     

Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1^ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.     

Multiple degradation pathways for misfolded mutants of the yeast plasma membrane ATPase, Pma1

To understand protein sorting and quality control in the secretory pathway, we have analyzed intracellular trafficking of the yeast plasma membrane ATPase, Pma1. Pma1 is ideal for such studies because it is a very abundant polytopic membrane protein, and its localization and activity at the plasma membrane are essential for cell viability and growth. We have tested whether the cytoplasmic amino- and carboxyl-terminal domains of Pma1 carry sorting information. As the sole copy of Pma1, mutants truncated at either NH2 or COOH termini are targeted at least partially to the plasma membrane and have catalytic activity to sustain cell viability. The mutants are also delivered to degradative pathways. Strikingly, NH2- and COOH-terminal Pma1 mutants are differentially recognized for degradation at distinct cellular locales. COOH-terminal mutants are recognized for destruction by endoplasmic reticulum-associated degradation. By contrast, NH2-terminal mutants escape detection by endoplasmic reticulum-associated degradation entirely, and undergo endocytosis for vacuolar degradation after apparently normal cell surface targeting. Both NH2- and COOH-terminal mutants are conformationally abnormal, as revealed by increased sensitivity to tryptic cleavage, but are able to assemble to form oligomers. We propose that different quality control mechanisms may assess discrete domains of Pma1 rather than a global conformational state.     

Partial Purification and Characterization of An ATPase in Mung Bean Hypocotyl Plasma Membrane: Suggestion for A New Type of Higher Plant Plasma Membrane ATPase

A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg^{2 +} was required for the enzyme activity, whereasK⁺, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-³²P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [¹⁴C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. ¹Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)     

In vivo analysis of Saccharomyces cerevisiae plasma membrane ATPase Pma1p isoforms with increased in vitro H+/ATP stoichiometry

Plasma membrane H⁺-ATPase isoforms with increased H⁺/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H⁺/ATP stoichiometries was analysed by measuring biomass yields of anaerobic maltose-limited chemostat cultures expressing only the different PMA1 alleles. In vivo H⁺/ATP stoichiometries of wildtype Pma1p and the two isoforms did not differ significantly.     

Yeast Phosphofructokinase-1 Subunit Pfk2p Is Necessary for pH Homeostasis and Glucose-dependent Vacuolar ATPase Reassembly

V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V₁ domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed.     

Molecular Determinants that Regulate Plasma Membrane Association of HIV-1 Gag

Human immunodeficiency virus type 1 assembly is a multistep process that occurs at the plasma membrane (PM). Targeting and binding of Gag to the PM are the first steps in this assembly process and are mediated by the matrix domain of Gag. This review highlights our current knowledge on viral and cellular determinants that affect specific interactions between Gag and the PM. We will discuss potential mechanisms by which the matrix domain might integrate three regulatory components, myristate, phosphatidylinositol-(4,5)-bisphosphate, and RNA, to ensure that human immunodeficiency virus type 1 assembly occurs at the PM.     

Localization of a Mg2+-Activated ATPase in the Plasma Membrane of Trypanosoma cruzi1

The Wachstein and Meisel incubation medium was used to detect ATPase activity in epimastigote, spheromastigote (amastigote), and bloodstream trypomastigote forms of Trypanosoma cruzi. Reaction product, indicative of enzyme activity, was associated with the plasma membrane covering the cell body and the flagellum of the parasite. No reaction product was found in the portion of the plasma membrane lining the flagellar pocket. The plasma membrane-associated ATPase activity was not inhibited by ouabain or oligomycin, was detected in incubation medium without K⁺, was inhibited by prolonged glutaraldehyde fixation, and its activity was diminished when Mg²⁺ was omitted from the incubation medium. The Ernst medium was used to detect Na⁺-K⁺-ATPase activity in T. cruzi. No reaction product indicative of the presence of this enzyme was detected. Reaction product indicative of 5'-nucleotidase was not detected in T. cruzi. Acid phosphatase activity was detected in lysosomes. These results indicate that a Mg²⁺-activated ATPase is present in the plasma membrane of T. cruzi and that it can be used as an enzyme marker, provided that the mitochondrial and flagellar ATPases are inhibited, to assess the purity of plasma membrane fractions isolated from this parasite.