KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion

KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.     

Organelle membrane fusion: a novel function for the syntaxin homolog Ufe1p in ER membrane fusion

The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane. Instead, ER membranes require a NSF-related ATPase, Cdc48p. Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated. We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs. We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.     

Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (. Genetics. 142:393-406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE-associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.     

Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins

We have identified and characterized a 9S protein complex from a Xenopus ovary cytosolic subfraction (fraction A) that constitutes this fraction's activity in recognizing a model nuclear import substrate and docking it at the nuclear pore complex. Because of its function, the complex is termed karyopherin. The 54- and 56-kDa subunits of the complex are termed alpha 1 and alpha 2, respectively, and the 97-kDa subunit is termed beta. In an alternative approach we have identified karyopherin beta from a rat liver cytosolic subfraction A by using immobilized rat nucleoporin Nup98 in a single, affinity-based enrichment step. We have molecularly cloned and sequenced rat karyopherin beta. Comparison with protein sequence data banks showed no significant similarity to other known proteins. Using nitrocellulose-immobilized rat liver nuclear envelope proteins and nuclear import substrate as a ligand, we found Xenopus fraction A-dependent binding to at least three bona fide nucleoporins (Nup214, Nup153, and Nup98) and to a candidate nucleoporin with an estimated molecular mass of 270 kDa. We propose that these nucleoporins function as docking proteins for karyopherin-mediated binding of substrate in a nuclear import/export pathway across the nuclear pore complex.     

The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function

Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.     

Signal sequence recognition in posttranslational protein transport across the yeast ER membrane

We have analyzed how the signal sequence of prepro-alpha-factor is recognized during the first step of posttranslational protein transport into the yeast endoplasmic reticulum. Cross-linking studies indicate that the signal sequence interacts in a Kar2p- and ATP-independent reaction with Sec61p, the multispanning membrane component of the protein-conducting channel, by intercalation into transmembrane domains 2 and 7. While bound to Sec61p, the signal sequence forms a helix that is contacted on one side by Sec62p and Sec71p. The binding site is located at the interface of the protein channel and the lipid bilayer. Signal sequence recognition in cotranslational translocation in mammals appears to occur similarly. These results suggest a general mechanism by which the signal sequence could open the channel for polypeptide transport.     

N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.     

Nuf2, a spindle pole body-associated protein required for nuclear division in yeast

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.     

Formation and properties of S-protein complex with S-peptide-containing fusion protein

A fusion protein (FP) comprised of the RNase A S-peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S-protein at a stoichiometric ratio 1:1 with Kdiss = 5.0 x 10(-7) M. The S-protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S-protein by RNase activity assays. The possibility of effective purification of S-peptide-containing proteins by affinity chromatography on an S-protein-Sepharose column has been demonstrated.     

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.     

Oligomerization-dependent folding of the membrane fusion protein of Semliki Forest virus

The spikes of alphaviruses are composed of three copies of an E2-E1 heterodimer. The E1 protein possesses membrane fusion activity, and the E2 protein, or its precursor form, p62 (sometimes called PE2), controls this function. Both proteins are, together with the viral capsid protein, translated from a common C-p62-E1 coding unit. In an earlier study, we showed that the p62 protein of Semliki Forest virus (SFV) dimerizes rapidly and efficiently in the endoplasmic reticulum (ER) with the E1 protein originating from the same translation product (so-called heterodimerization in cis) (B.-U. Barth, J. M. Wahlberg, and H. Garoff, J. Cell Biol. 128:283-291, 1995). In the present work, we analyzed the ER translocation and folding efficiencies of the p62 and E1 proteins of SFV expressed from separate coding units versus a common one. We found that the separately expressed p62 protein translocated and folded almost as efficiently as when it was expressed from a common coding unit, whereas the independently expressed E1 protein was inefficient in both processes. In particular, we found that the majority of the translocated E1 chains were engaged in disulfide-linked aggregates. This result suggests that the E1 protein needs to form a complex with p62 to avoid aggregation. Further analyses of the E1 aggregation showed that it occurred very rapidly after E1 synthesis and could not be avoided significantly by the coexpression of an excess of p62 from a separate coding unit. These latter results suggest that the p62-E1 heterodimerization has to occur very soon after E1 synthesis and that this is possible only in a cis-directed reaction which follows the synthesis of p62 and E1 from a common coding unit. We propose that the p62 protein, whose synthesis precedes that of the E1 protein, remains in the translocon of the ER and awaits the completion of E1. This strategy enables the p62 protein to complex with the E1 protein immediately after the latter has been made and thereby to control (suppress) its fusion activity.     

The precore sequence of hepatitis B virus is required for nuclear localization of the core protein

The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.     

A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae

Calcitonin, the serum calcium-lowering hormone, has been used in the treatment of hypercalcemia of malignancy and postmenopausal osteoporosis in humans for several years without any adverse effects. Recent studies in rats have indicated that calcitonin may be associated with morphologic effects on the pituitary. A large study was performed on 2 strains of rats, Sprague-Dawley (SD) and Fischer-344 (F-344), with 2 types of calcitonin, salmon-derived (sCT) and porcine-derived (pCT) calcitonin to evaluate possible effects on the pituitary. Sixteen groups of 42 male and 42 female SD or F-344 rats were given 0 (vehicle control), 1.25, 5.0, or 80.0 IU/kg/day of sCT or pCT, once daily, subcutaneously, for 1 yr. An increased incidence of adenomas of the adenohypophysis was observed in male SD rats at all dose levels of sCT, female SD rats given 80 IU/kg/day of sCT, male SD rats at the high dose level of pCT, and male F-344 rats at the high dose level of sCT. Also, an increased incidence of total proliferative lesions, due mostly to an increased incidence of focal hyperplasia of the pars distalis, occurred in female F-344 rats given the high dose of sCT. These pituitary proliferations were histologically similar to those that occur spontaneously, and the incidences observed were comparable to those that could occur in rats on 2-yr or lifetime studies, indicating that the injection of calcitonin had decreased the latency period.     

Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors alpha-, beta-, and gamma-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant alpha-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in alpha-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.     

Evidence that phospholipase A2 activity is required for Golgi complex and trans Golgi network membrane tubulation

Membrane tubules of uniform diameter (60-80 nm) and various lengths (up to several micrometers) emanate from elements of the Golgi stack and trans Golgi network (TGN). These organelle membrane tubules are thought to be involved in membrane trafficking and maintenance of Golgi/TGN architecture. The number of these tubules, and their frequency of formation, can be greatly enhanced by the fungal metabolite brefeldin A (BFA), an inhibitor of Golgi/TGN-associated coated vesicle formation. We show here that BFA stimulation of Golgi and TGN membrane tubulation, and the resultant retrograde transport of resident Golgi enzymes to the endoplasmic reticulum, was potently inhibited by a number of membrane-permeant antagonists of phospholipase A2 (PLA2; EC 3.1.1.4) activity. In addition, PLA2 inhibitors on their own caused a reversible fragmentation of the Golgi complex into juxtanuclear, stacked cisternal elements. We conclude from these observations that tubulation of Golgi complex and TGN membranes requires a PLA2 activity, and that this activity may participate not only in Golgi tubule-mediated retrograde trafficking to the endoplasmic reticulum, but also in the maintenance of Golgi complex architecture.     

Functional conservation of cytosolic proteins required for endosomal vesicle fusion

Recent studies suggest that intracellular membrane traffic relies upon families of related proteins which confer specificity to individual transport reactions but which operate in tandem with a ubiquitous fusogenic complex containing the N-ethylmaleimide-sensitive fusion protein (NSF). The extent to which components of this process are functionally conserved is apparent from the finding that yeast Sec18 protein (Sec18p) can substitute or mammalian NSF in intra-Golgi transport reactions. Here we report that yeast cytosol can support mammalian endosomal vesicle fusion, demonstrating conservation of cytosolic components required for this reaction. Furthermore, under conditions in which the fusion reaction is NSF-dependent we show that yeast Sec18p can functionally substitute for NSF, showing that the yeast protein is capable of catalysing at least two distinct mammalian membrane fusion events. In addition we exploit the complex pattern of sensitivity of the mammalian reaction to N-ethylmaleimide (NEM), coupled with the use of yeast cytosol, to dissect a number of factors required for fusion. We reveal at least three novel NEM-sensitive activities. One of these can be restored by yeast cytosol suggesting that it is functionally conserved.