Saccharomyces cerevisiae Lacking Btn1p Modulate Vacuolar ATPase Activity to Regulate pH Imbalance in the Vacuole

Abstract

The vacuolar H ؉ -ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V 0 and V 1 V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-⌬), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-⌬ is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-⌬ at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.The neuronal ceroid-lipofuscinoses (NCLs) 2 are the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births (1, 2). The NCL disorders are inherited in an autosomal recessive manner, with mutations in seven distinct genes resulting in pathologically similar disease with a different age of onset (3, 4). The NCLs are characterized by the accumulation of autofluorescent hydrophobic material in the lysosomes of neurons, and to lesser extent, other cell types (5, 6); however, the molecular basis behind this storage and the disease remains unknown. The juvenile form of NCL results from mutations in the CLN3 gene, which codes for a lysosomal transmembrane protein (7-9).We have previously reported that the Saccharomyces cerevisiae BTN1 gene product has high sequence similarity with the human CLN3 gene product. BTN1 encodes a non-essential protein that is 39% identical and 59% similar to human CLN3 (10). Studies have revealed that Btn1p is located in the vacuolar membrane (11, 12) and, although the primary function of this protein remains unclear, it has been implicated in several cellular pathways. Lack of BTN1 resulted in resistance to D-(Ϫ)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), and this phenotype is complemented by expression of human Cln3p, indicating that yeast Btn1p and human Cln3p likely have a conserved function (13). Resistance of btn1-⌬...

Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast

“…As in previous studies (26,52), we find that the cytosolic pH of cells supplied with glucose is relatively insensitive to extracellular pH, ranging from 6.7 at pH ext 5 to pH 7.1 at pH ext ϭ 7.5, and is even more constant if KCl is present (cytosolic pH 7.0 -7.1). Vacuolar pH appears to be more dependent on extracellular conditions (20,30) but is generally higher in yeast vacuoles than in mammalian lysosomes. We propose that the rise in cytosolic pH upon glucose addition reflects both the availability of metabolically generated ATP and H ϩ and glu-4 G. A. Martínez-Muñ oz and P. Kane, unpublished data.…”

“…V-ATPases are also activated by glucose, but they reversibly disassemble in response to glucose deprivation and readdition, resulting in higher levels of assembled pumps and ATPase activity at the vacuole in the presence of glucose (18,19). The activity of both pumps is sensitive to pH, indicating that they may be tuned to respond to changes in cytosolic pH in vivo (20,21). Pma1p, in particular, is activated in response to cytosolic acidification (21), and this mode of regulation is likely to be important for maintenance of cytosolic pH in a narrow range, relatively independent of extracellular pH.…”

The fission yeast model for the lysosomal storage disorder Batten disease predicts disease severity caused by mutations in CLN3

“…Despite much effort since its identification in 1995, the function of CLN3 has remained elusive, although it too has been implicated in multiple roles, including lysosome homeostasis (Holopainen et al, 2001;Ramirez-Montealegre and Pearce, 2005;Pohl et al, 2007), autophagy (Cao et al, 2006), cytoskeletal organisation (Luiro et al, 2004;Luiro et al, 2006) and lipid synthesis or modification (Narayan et al, 2006;Hobert and Dawson, 2007). Work in other organisms, especially Saccharomyces cerevisae, also supports a role for CLN3/Btn1p in vacuole pH homeostasis (Pearce and Sherman, 1998;Pearce et al, 1999a;Pearce et al, 1999b;Pearce and Sherman, 1999;Chattopadhyay et al, 2003;Kim et al, 2003;Padilla-Lopez and Pearce, 2006). Significantly, expression of CLN3 restored phenotypes arising from deletion of btn1 in these organisms, indicating that these proteins exert a similar basic function.…”

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…Our activity assay and the use of inhibitors was based on (Serrano 1978;Clelland and Saleuddin 2000;Lunde and Kubo 2000;Padilla-Lopez and Pearce 2006), with modifications, as follows. The assay mixture contained the activity buffer (50 mM MES/TRIS, 5 mM MgCl 2 , pH 7.0), 5 mM sodium azide (to inhibit mitochondrial ATPase), 0.2 mM ammonium molybdate (to inhibit acid phosphatases), 100 μM sodium orthovanadate (to inhibit plasma membrane proton-ATPase).…”

“…Since the substrate of the reaction is MgATP and MgCl 2 is in excess to Na 2 ATP, the substrate concentration was close to 2 mM (at least in the beginning of the reaction). Several inhibitors that are known to inhibit other ATPases (Serrano 1978;Clelland and Saleuddin 2000;Lunde and Kubo 2000;Padilla-Lopez and Pearce 2006) were tested. These 'other' inhibitors removed only about 20% of the total ATPase activity, even at relatively high inhibitor concentration.…”