The phosphoinositide phosphatase Sac1p controls trafficking of the yeast Chs3p chitin synthase.

Phosphoinositide phosphatases play an essential but as yet not well-understood role in lipid-based signal transduction. Members of a subfamily of these enzymes share a specific domain that was first identified in the yeast Sac1 protein [1]. Sac1 homology domains were shown to exhibit 3- and 4-phosphatase activity in vitro [2, 3] and were also found, in addition to rat and yeast Sac1p, in yeast Inp/Sjl proteins [4, 5] and mammalian synaptojanins [6]. Despite the detailed in vitro characterization of the enzymatic properties of yeast Sac1p, the exact cellular function of this protein has remained obscure. We report here that Sac1p has a specific role in secretion and acts as an antagonist of the phosphatidylinositol 4-kinase Pik1p in Golgi trafficking. Elimination of Sac1p leads to excessive forward transport of chitin synthases and thus causes specific cell wall defects. Similar defects in membrane trafficking are caused by the overexpression of PIK1. Taken together, these findings provide strong evidence that the generation of PtdIns(4)P is sufficient to trigger forward transport from the Golgi to the plasma membrane and that Sac1p is critically required for the termination of this signal.

Reconstitution of yeast microsomal lipid flip-flop using endogenous aminophospholipids.

The molecular basis of transbilayer movement or flipping of phospholipids in the endoplasmic reticulum is largely unknown. To circumvent the problems inherent to studies with artificial phospholipid analogs, we studied microsomal flip-flop of endogenous phosphatidylethanolamine in yeast. The transbilayer transport of phosphatidylethanolamine was measured in reconstituted proteoliposomes derived from microsomal detergent extracts. Our results demonstrate that flipping is protease sensitive but does not require metabolic energy. Our assay is the first to use the endogenous substrate of the so-called 'flippase' to study phospholipid translocation in endomembranes and may therefore be crucial for the understanding of the catalytic properties of this elusive enzyme.

Sac1p plays a crucial role in microsomal ATP transport, which is distinct from its function in Golgi phospholipid metabolism.

Analysis of microsomal ATP transport in yeast resulted in the identification of Sac1p as an important factor in efficient ATP uptake into the endoplasmic reticulum (ER) lumen. Yet it remained unclear whether Sac1p is the authentic transporter in this reaction. Sac1p shows no homology to other known solute transporters but displays similarity to the N-terminal non-catalytic domain of a subset of inositol 5'-phosphatases. Furthermore, Sac1p was demonstrated to be involved in inositol phospholipid metabolism, an activity whose absence contributes to the bypass Sec14p phenotype in sac1 mutants. We now show that purified recombinant Sac1p can complement ATP transport defects when reconstituted together with sac1Delta microsomal extracts, but is unable to catalyze ATP transport itself. In addition, we demonstrate that sac1Delta strains are defective in ER protein translocation and folding, which is a direct consequence of impaired ATP transport function and not related to the role of Sac1p in Golgi inositol phospholipid metabolism. These data suggest that Sac1p is an important regulator of microsomal ATP transport providing a possible link between inositol phospholipid signaling and ATP-dependent processes in the yeast ER.

Essential role for diacylglycerol in protein transport from the yeast Golgi complex.

Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway). Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin, a protein found in rat brain. Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi. Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia coli further impairs it. The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles.

Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation.

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro-alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.

Effects of ATP on ligand recognition of platelet fibrinogen receptor on GPIIb-IIIa.

The recent discovery of 8-azido-ATP binding sites on the platelet fibrinogen receptor glycoprotein complex GPIIb-IIIa suggests that extracellular ATP may directly modulate function of GPIIb-IIIa. In this study we investigated the effect of ATP on ligand binding to GPIIb-IIIa. Fibrinogen-mediated aggregation of washed platelets was inhibited by ATP and 8-azido-ATP in a dose-dependent manner, independent of the agonist (thrombin, collagen, epinephrine, phorbol 12-myristate 13-acetate) used to induce platelet activation. In addition, 8-azido-ATP and ATP inhibited binding of 125I-labeled fibrinogen to thrombin- and phorbol ester-activated platelets. Interaction of nonstimulated platelets with solid-phase fibrinogen was also reduced by 8-azido-ATP and ATP. Moreover, fibrinogen mimetic peptide-induced conformational change of GPIIb-IIIa on resting platelets was reduced in the presence of both nucleotides. Finally, photoincorporation of 8-azido-[gamma-32P]ATP into GPIIb-IIIa was suppressed by GRGDSP but not by the biologically inactive GRGESP peptide. Thus interaction of ATP with 8-azido-ATP binding sites present on GPIIb-IIIa modulate receptor function, which may play a role in regulation of in vivo platelet aggregation.

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.

Labeling of two different regions of the nucleotide binding site of the uncoupling protein from brown adipose tissue mitochondria with two ATP analogs.

The nucleotide binding site of the uncoupling protein (UCP) from brown adipose tissue was mapped by photoaffinity labeling with 2-azidoadenosine 5'-triphosphate (2-azido-ATP) and by affinity labeling with 3'-O-(5-fluoro-2,4-dinitrophenyl)adenosine 5'-triphosphate (FDNP-ATP). Both analogs bind with high affinity and specificity to the UCP in intact mitochondria, as well as to the isolated solubilized protein. Reversible binding at 4 degrees C in the dark is competitively blocked by GTP. Like the natural ligands ATP and GTP, both analogs are capable of inhibiting the H+/OH- conductance of the UCP as measured in proteoliposomes with reconstituted UCP. 2-azido-ATP was incorporated into UCP in mitochondria in the presence of carboxyatractylate, while FDNP-ATP was inserted into isolated UCP by prolonged incubation at room temperature under pH variation. Both reactions can be blocked by GTP. The incorporation of 2-azido-ATP could be localized between residues 258 and 283 by cleavage with CNBr. Solid-phase sequencing of the homoserine-linked radioactive peptide indicated that the 2-azido-ATP was linked to threonine-263. The incorporation of FDNP-ATP could be assigned by cleavage with CNBr and alternatively with trypsin at a locus of covalent attachment between residues 238 and 255. On the basis of published data that no tyrosine participates in nucleotide binding of the UCP, the probable residue reacting with FDNP-ATP is cysteine-253.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP.

The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-induced conformational change of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3). Photoaffinity labeling of intact platelets with 8-azido-[gamma-32P]ATP exclusively modifies two plasma-membrane glycoproteins which are identical with both subunits of GPIIb-IIIa. The presence of adenine-nucleotide-binding sites on GPIIb-IIIa implies that the platelet fibrinogen receptor is directly regulated by extracellular adenine nucleotides.

A microsomal protein is involved in ATP-dependent transport of presecretory proteins into mammalian microsomes.

Ribonucleoparticle (i.e. ribosome and SRP)-independent transport of proteins into mammalian microsomes is stimulated by a cytosolic ATPase which involves proteins belonging to the hsp70 family. Here we addressed the question of whether there are additional nucleoside triphosphate requirements involved in this transport mechanism. We employed a purified presecretory protein which upon solubilization in dimethyl sulfoxide and subsequent dilution into an aqueous buffer was processed by and transported into mammalian microsomes in the absence of the cytosolic ATPase. Membrane insertion of this precursor protein was found to depend on the hydrolysis of ATP and to involve a microsomal protein which can be photoaffinity inactivated with azido-ATP. Furthermore, a microsomal protein with a similar sensitivity towards photoaffinity modification with azido-ATP was observed to be involved in ribonucleoparticle-dependent transport. We suggest that a novel microsomal protein which depends on ATP hydrolysis is involved in membrane insertion of both ribonucleoparticle-dependent and -independent precursor proteins.

Photoaffinity labeling of dog pancreas microsomes with 8-azido-ATP inhibits association of nascent preprolactin with the signal sequence receptor complex.

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido-ATP and which is distinct from the ATP-binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl-86mer, was employed. Here we show that the nascent preprolactin did not become associated with the alpha-subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido-ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido-ATP acts prior to the signal sequence receptor complex.

The ADP/ATP carrier from yeast (AAC-2) is uniquely suited for the assignment of the binding center by photoaffinity labeling.

The ADP/ATP carrier from yeast was photoaffinity-labeled in mitochondria with 2-azido-[alpha-32P]ATP in a binding-center-specific, i.e. carboxyatractylate-sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC-2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115-210 contained all the incorporated label which corresponds to the second domain within the triple-domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171-Gly 172 site, all the label was found in the C-terminal 16 kDa fragment. Thus the 2-azido-ATP incorporation is clearly delimited to the 172-210 segment. 8-Azido-[alpha-32P]ATP could be site-specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2-azido-ATP. The label was also exclusively found in the 172-210 region. With both forms no incorporation into the C-terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.