Cloning and functional characterization of a subunit of the transporter associated with antigen processing

The transporter associated with antigen processing (TAP) is essential for the transport of antigenic peptides across the membrane of the endoplasmic reticulum. In addition, TAP interacts with major histocompatibility complex class I heavy chain (HC)/β2-microglobulin (β2-m) dimers. We have cloned a cDNA encoding a TAP1/2-associated protein (TAP-A) corresponding in size and biochemical properties to tapasin, which was recently suggested to be involved in class I–TAP interaction (Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T. & Cresswell, P. (1996) Immunity 5, 103–114). The cDNA encodes a 448-residue-long ORF, including a signal peptide. The protein is predicted to be a type I membrane glycoprotein with a cytoplasmic tail containing a double-lysine motif (-KKKAE-COOH) known to maintain membrane proteins in the endoplasmic reticulum. Immunoprecipitation with anti-TAP1 or anti-TAP-A antisera demonstrated a consistent and stoichiometric association of TAP-A with TAP1/2. Class I HC and β2-m also were coprecipitated with these antisera, indicating the presence of a pentameric complex. In pulse–chase experiments, class I HC/β2-m rapidly dissociated from TAP1/2-TAP-A. We propose that TAP is a trimeric complex consisting of TAP1, TAP2, and TAP-A that interacts transiently with class I HC/β2-m. In peptide-binding assays using cross-linkable peptides and intact microsomes, TAP-A bound peptides only in the presence of ATP whereas binding of peptides to TAP1/2 was ATP-independent. This suggests a direct role of TAP-A in peptide loading onto class I HC/β2-m dimer.

Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains

Incubation of Na/K-ATPase with ascorbate plus H2O2 produces specific cleavage of the α subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The β subunit is not cleaved. Cleavages depend on the presence of contaminant or added Fe2+ ions, as inferred by suppression of cleavages with nonspecific metal complexants (histidine, EDTA, phenanthroline) or the Fe3+-specific complexant desferrioxamine, or acceleration of cleavages by addition of low concentrations of Fe2+ but not of other heavy metal ions. Na/K-ATPase is inactivated in addition to cleavage, and both effects are insensitive to OH⋅ radical scavengers. Cleavages are sensitive to conformation. In low ionic strength media (E2) or media containing Rb ions [E2(Rb)], cleavage is much faster than in high ionic strength media (E1) or media containing Na ions (E1Na). N-terminal fragments and two C-terminal fragments (N-terminals E214 and V712) have been identified by amino acid sequencing. Approximate positions of other cleavages were determined with specific antibodies. The results suggest that Fe2+ (or Fe3+) ions bind with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the α subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations but move apart in the E1 or E1Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.

Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY). In E. coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain) with an α-helical domain on its N terminus (N domain). The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques. Methods to describe the reaction kinetically are presented. The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases. The intrinsic guanine nucleotide dissociation rates of FtsY are about 105 times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor. Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase–nucleotide exchange factor complex not only in its structure but also kinetically. The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor. From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

TnTIN and TnTAP: Mini-transposons for site-specific proteolysis in vivo

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.

PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer’s disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.

Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I

N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.

Acylation stabilizes a protease-resistant conformation of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protein acylation is an important way in which a number of proteins with a variety of functions are modified. The physiological role of the acylation of cellular proteins is still poorly understood. Covalent binding of fatty acids to nonintegral membrane proteins is thought to produce transient or permanent enhancement of the association of the polypeptide chains with biological membranes. In this paper, we investigate the functional role for the palmitoylation of an atypical membrane-bound protein, yeast protoporphyrinogen oxidase, which is the molecular target of diphenyl ether-type herbicides. Palmitoylation stabilizes an active heat- and protease-resistant conformation of the protein. Palmitoylation of protoporphyrinogen oxidase has been demonstrated to occur in vivo both in yeast cells and in a heterologous bacterial expression system, where it may be inhibited by cerulenin leading to the accumulation of degradation products of the protein. The thiol ester linking palmitoleic acid to the polypeptide chain was shown to be sensitive to hydrolysis by hydroxylamine and also by the widely used serine-protease inhibitor phenylmethylsulfonyl fluoride.

Visualization of a peripheral stalk in V-type ATPase: Evidence for the stator structure essential to rotational catalysis

F- and V-type ATPases are central enzymes in energy metabolism that couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes. They consist of a soluble headpiece that contains the catalytic sites and an integral membrane-bound part that conducts the ion flow. Energy coupling is thought to occur through the physical rotation of a stalk that connects the two parts of the enzyme complex. This mechanism implies that a stator-like structure prevents the rotation of the headpiece relative to the membrane-bound part. Such a structure has not been observed to date. Here, we report the projected structure of the V-type Na+-ATPase of Clostridium fervidus as determined by electron microscopy. Besides the central stalk, a second stalk of 130 Å in length is observed that connects the headpiece and membrane-bound part in the periphery of the complex. This additional stalk is likely to be the stator.

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

The Yck2 Yeast Casein Kinase 1 Isoform Shows Cell Cycle-specific Localization to Sites of Polarized Growth and Is Required for Proper Septin Organization

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP–Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP–Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.