Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent γ-glutamyl carboxylation during protein processing and block the secretion of under-γ-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-γ-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FIIRHH and FIIHRR, FIIRRH and FIIHHR, FIIRHR and FIIHRH) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-γ-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FIIHrhH and FIIHhrH, FIIRrhR, and FIIRhrR) was used to determine that the first of the two kringles plays a more important role in the recognition process.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (β2-microglobulin, β2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and β2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous β2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and β2m are independent events. The exchange rate of β2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous β2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

Bimodal regulation of Na+–Ca2+ exchanger by β-adrenergic signaling pathway in shark ventricular myocytes

In shark heart, the Na+–Ca2+ exchanger serves as a major pathway for both Ca2+ influx and efflux, as there is only rudimentary sarcoplasmic reticulum in these hearts. The modulation of the exchanger by a β-adrenergic agonist in whole-cell clamped ventricular myocytes was compared with that of the Na+–Ca2+ exchanger blocker KB-R7943. Application of 5 μM isoproterenol and 10 μM KB-R7943 suppressed both the inward and the outward Na+–Ca2+ exchanger current (INa−Ca). The isoproterenol effect was mimicked by 10 μM forskolin. Isoproterenol and forskolin shifted the reversal potential (Erev) of INa−Ca by approximately −23 mV and −30 mV, respectively. An equivalent suppression of outward INa−Ca by KB-R7943 to that by isoproterenol produced a significantly smaller shift in Erev of about −4 mV. The ratio of inward to outward exchanger currents was also significantly larger in isoproterenol- than in control- and KB-R7943-treated myocytes. Our data suggest that the larger ratio of inward to outward exchanger currents as well as the larger shift in Erev with isoproterenol results from the enhanced efficacy of Ca2+ efflux via the exchanger. The protein kinase A-mediated bimodal regulation of the exchanger in parallel with phosphorylation of the Ca2+ channel and enhancement of its current may have evolved to satisfy the evolutionary needs for accelerated contraction and relaxation in hearts of animals with vestigial sarcoplasmic Ca2+ release stores.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Calmodulin kinase is a molecular switch for cardiac excitation –contraction coupling

Signaling between cell membrane-bound L-type Ca2+ channels (LTCC) and ryanodine receptor Ca2+ release channels (RyR) on sarcoplasmic reticulum (SR) stores grades excitation–contraction coupling (ECC) in striated muscle. A physical connection regulates LTCC and RyR in skeletal muscle, but the molecular mechanism for coordinating LTCC and RyR in cardiomyocytes, where this physical link is absent, is unknown. Calmodulin kinase (CaMK) has characteristics suitable for an ECC coordinating molecule: it is activated by Ca2+/calmodulin, it regulates LTCC and RyR, and it is enriched in the vicinity of LTCC and RyR. Intact cardiomyocytes were studied under conditions where CaMK activity could be controlled independently of intracellular Ca2+ by using an engineered Ca2+-independent form of CaMK and a highly specific CaMK inhibitory peptide. CaMK reciprocally enhanced L-type Ca2+ current and reduced release of Ca2+ from the SR while increasing SR Ca2+ content. These findings support the hypothesis that CaMK is required to functionally couple LTCC and RyR during cardiac ECC.

Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

NMR structure of the calreticulin P-domain

The NMR structure of the rat calreticulin P-domain, comprising residues 189–288, CRT(189–288), shows a hairpin fold that involves the entire polypeptide chain, has the two chain ends in close spatial proximity, and does not fold back on itself. This globally extended structure is stabilized by three antiparallel β-sheets, with the β-strands comprising the residues 189–192 and 276–279, 206–209 and 262–265, and 223–226 and 248–251, respectively. The hairpin loop of residues 227–247 and the two connecting regions between the β-sheets contain a hydrophobic cluster, where each of the three clusters includes two highly conserved tryptophyl residues, one from each strand of the hairpin. The three β-sheets and the three hydrophobic clusters form a repeating pattern of interactions across the hairpin that reflects the periodicity of the amino acid sequence, which consists of three 17-residue repeats followed by three 14-residue repeats. Within the global hairpin fold there are two well-ordered subdomains comprising the residues 219–258, and 189–209 and 262–284, respectively. These are separated by a poorly ordered linker region, so that the relative orientation of the two subdomains cannot be precisely described. The structure type observed for CRT(189–288) provides an additional basis for functional studies of the abundant endoplasmic reticulum chaperone calreticulin.

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

Selective Formation of Sed5p-containing SNARE Complexes Is Mediated by Combinatorial Binding Interactions

Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE–SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.

Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic EventsV⃞

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocity Vmax of 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.

Excitation–contraction uncoupling by a human central core disease mutation in the ryanodine receptor

Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca2+-induced damage stemming from mutant RyR1 proteins forming “leaky” sarcoplasmic reticulum (SR) Ca2+ release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164–4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca2+ release without significantly altering resting cytosolic Ca2+ levels, SR Ca2+ content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca2+ release, again without altering resting cytosolic Ca2+ levels, SR Ca2+ content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca2+ release, rather than the expression of overactive or leaky SR Ca2+ release channels.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Identification of a Calmodulin-Regulated Ca2+-ATPase in the Endoplasmic Reticulum1

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical “ER-type” Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.