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Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-Dflopreceptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-Dflipundergoes complex glycosylation and reaches the plasma membrane at >10× higher levels than GluR-Dflop, as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-Dflopnear to the level exhibited by GluR-Dflip. Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.

Oxysterol binding protein (OSBP) homologs comprise a family of 12 proteins in humans ( Jaworski et al., 2001 ; Lehtoet al., 2001 ). Two variants of OSBP-related protein (ORP) 1 have been identified: a short one that consists of the carboxy-terminal ligand binding domain only (ORP1S, 437 aa) and a longer N-terminally extended form (ORP1L, 950 aa) encompassing three ankyrin repeats and a pleckstrin homology domain (PHD). We now report that the two mRNAs show marked differences in tissue expression. ORP1S predominates in skeletal muscle and heart, whereas ORP1L is the most abundant form in brain and lung. On differentiation of primary human monocytes into macrophages, both ORP1S and ORP1L mRNAs were induced, the up-regulation of ORP1L being >100-fold. The intracellular localization of the two ORP1 variants was found to be different. Whereas ORP1S is largely cytosolic, the ORP1L variant localizes to late endosomes. A significant amount of ORP1S but only little ORP1L was found in the nucleus. The ORP1L ankyrin repeat region (aa 1–237) was found to localize to late endosomes such as the full-length protein. This localization was even more pronounced for a fragment that additionally includes the PHD (aa 1–408). The amino-terminal region of ORP1L consisting of the ankyrin repeat and PHDs is therefore likely to be responsible for the targeting of ORP1L to late endosomes. Interestingly, overexpression of ORP1L was found to enhance the LXRα-mediated transactivation of a reporter gene, whereas ORP1S failed to influence this process. The results suggest that the two forms of ORP1 are functionally distinct and that ORP1L is involved in control of cellular lipid metabolism.

Bacterial biofilms impair the operation of many industrial processes. Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D. geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the D. geothermalis E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living D. geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment. D. geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47. Deinococcus radiodurans strain DSM 20539 T also formed tenacious biofilms. This paper shows that D. geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.

Cholesterol entering cells in low-density lipoproteins (LDL) via receptor-mediated endocytosis is transported to organelles of the late endocytic pathway for degradation of the lipoprotein particles. The fate of the free cholesterol released remains poorly understood, however. Recent observations suggest that late-endosomal cholesterol sequestration is regulated by the dynamics of lysobisphosphatidic acid (LBPA)-rich membranes [1]. Genetic studies have pinpointed a protein, Niemann–Pick C-1 (NPC-1), that is required for the mobilization of late-endosomal/lysosomal cholesterol by an unknown mechanism [2]. Here, we report the removal of accumulated cholesterol by overexpression of the NPC-1 protein in NPC-1-deficient fibroblasts from patients with Niemann–Pick disease, and in normal fibroblasts upon release of a progesterone-induced block of cholesterol transport. We show that late-endosomal/lysosomal cholesterol mobilization is specifically inhibited by microinjection of Rab GDP-dissociation inhibitor (Rab-GDI). Moreover, clearance of the cholesterol deposits by NPC-1 in patients’ fibroblasts is accompanied by the redistribution of LBPA and of a lysosomal hydrolase that utilizes the mannose-6-phosphate receptor. Our results reveal, for the first time, the involvement of a specific molecular component of the membrane-trafficking machinery in cholesterol transport and the coupling of late-endosomal cholesterol egress to the trafficking of other lipid and protein cargo.

To investigate the distribution of lipids through the Golgi complex, we analyzed the envelopes of several viruses that assemble in different subcompartments of the Golgi, as well as subcellular fractions. Our results indicate that each Golgi subcompartment has a distinct phospholipid composition due mainly to differences in the relative amounts of semilysobisphosphatidic acid (SLBPA), sphingomyelin, phosphatidylserine, and phosphatidylinositol. Interestingly, SLBPA is enriched in the adjacent Golgi networks compared with the Golgi stack, and this enrichment varies with cell type. The heterogeneous distribution of SLBPA through the Golgi complex suggests it may play an important role in the structure and/or function of this organelle.

A number of cellular proteins and viral spike proteins are cleaved at a basic recognition sequence. To characterize the membrane traffic step at which this proteolysis occurs we have studied the intracellular processing site of Semliki Forest virus (SFV) spike precursor p62 in BHK21 cells. The p62 is endoproteolytically cleaved at a tetrabasic Arg-His-Arg-Arg recognition sequence. Previously, it has been shown that the SFV p62 remains uncleaved when accumulated to the trans-Golgi network (TGN/20 degrees C block site). We show here that exit from the trans-Golgi is required for the cleavage of p62. Proteolytic processing was inhibited in synchronized assays when the 20 degrees C transport block was released in the presence of brefeldin A, energy inhibitors (azide and deoxyglucose; carbonyl cyanide m-chlorophenylhydrazone, CCCP) or an effector of trimeric G proteins, AlFn. Endocytosed antibodies against the SFV spike glycoproteins or antibodies against a peptide corresponding to the enzymatically active motif of furin inhibited cleavage of p62 at a post-TGN location. The results indicate a post-TGN communication step between exocytic and endocytic elements. Kinetic experiments suggested that this communication may involve an early compartment of the endocytic pathway.

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

The storage of glucose as glycogen in skeletal muscle is frequently impaired in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their nondiabetic relatives. Despite an intensive search for candidate genes associated with NIDDM, no data have been available on the gene coding for the key enzyme of this pathway, glycogen synthase.Using a human complementary DNA probe, the restriction enzyme Xbal, and Southern blot analysis, we identified two polymorphic alleles, A1 and A2, in the glycogen synthase gene. The gene was localized to chromosome 19. The A1A2 or A2A2 genotype was found in 30 percent of 107 patients with NIDDM but in only 8 percent of 164 nondiabetic subjects without a family history of NIDDM (P0.001). The diabetic patients with the A2 allele had a stronger family history of NIDDM (P = 0.019), a higher prevalence of hypertension (P = 0.008), and a more severe defect in insulin-stimulated glucose storage (P = 0.001) than the diabetic patients with the A1 allele. The concentration of the glycogen synthase protein in biopsy specimens of skeletal muscle from the patients with the A2 allele was normal, however, suggesting that expression of the gene was unaltered. The Xbal polymorphism was due to a change of a single base in an intron.The Xbal polymorphism of the glycogen synthase gene identifies a subgroup of patients with NIDDM characterized by a strong family history of NIDDM, a high prevalence of hypertension, and marked insulin resistance.

Recent results have provided increasing evidence for the existence of an intermediate membrane compartment between the rough endoplasmic reticulum and the Golgi complex which seems to function in protein sorting and the regulation of membrane traffic in the early part of the exocytic pathway. Localization of resident marker proteins has shown that this compartment consists of both peripheral and central elements. The aim of the present review is to combine the data on the pre-Golgi compartment with previous ideas of membrane traffic at the ER-Golgi interface. We propose a model which describes how mobile, endosome-like elements of the pre-Golgi compartment function in the generation of the compositional and functional boundary between the widely distributed ER and the more centrally located Golgi stacks.

Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.

Here we used electrospray ionization mass spectrometry for quantitative determination of lipid molecular species in human fibroblasts and their plasma membrane incorporated into enveloped viruses. Both influenza virus selecting ordered domains and vesicular stomatitis virus (VSV) depleted of such domains [Scheiffele, P., et al. (1999) J. Biol. Chem. 274, 2038-2044] were analyzed. The major difference between influenza and VSV was found to be a marked enrichment of glycosphingolipids in the former. The effect of chronic cholesterol loading on viral lipid composition was studied in Niemann-Pick type C (NPC) fibroblasts. Both NPC-derived influenza and VSV virions contained increased amounts of cholesterol. Furthermore, polyunsaturated phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were enriched in NPC-derived virions at the expense of the monounsaturated ones. When normal fibroblasts were acutely loaded with cholesterol using cyclodextrin complexes, an adjustment toward increasingly unsaturated phospholipid species was observed, most clearly for phosphatidylcholine and sphingomyelin. Our results provide evidence that (1) glycosphingolipids are enriched in domains through which influenza virus buds, (2) chronic cholesterol accumulation increases the cholesterol content of both glycosphingolipid-enriched and intervening plasma membrane domains, and (3) an increase in membrane cholesterol content is accompanied by an increased level of polyunsaturated species of the major membrane phospholipids. We suggest that remodeling of phospholipids toward higher unsaturation may serve as both an acute and a long-term adaptive mechanism in human cellular membranes against cholesterol excess.

The Semliki Forest virus (SFV) glycoprotein precursor p62 is processed to the E2 and E3 during the transport from the trans-Golgi network (TGN) to the cell surface. We have studied the regulation of the membrane fusion machinery (Rab/N-ethylmaleimide (NEM)-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)–SNAP receptor) in this processing. Activation of the disassembly of this complex with recombinant NSF stimulated the cleavage of p62 in permeabilized cells. Inactivation of NSF with a mutant α-SNAP(L294A) or NEM treatment inhibited processing of p62. Rab GDP dissociation inhibitor inhibited the cleavage. Inactivation of NSF blocks the transport of SFV glycoproteins and vesicular stomatitis virus G-glycoprotein from the TGN membranes to the cell surface. The results support the conclusion that inhibition of membrane fusion arrests p62 in the TGN and prevents its processing by furin.

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15 degrees C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15 degrees C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nm-diameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.

The effect of the carboxylic ionophore monensin on the maturation of Uukuniemi virus, a bunyavirus, and the transport of its two membrane glycoproteins, G1 and G2, were studied in chicken embryo fibroblasts and baby hamster kidney cells. Virus maturation, which occurs in the Golgi complex (E. Kuismanen, K. Hedman, J. Saraste, and R. F. Pettersson, Mol. Cell. Biol. 2:1444-1458, 1982; E. Kuismanen, B. Bång, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984), was effectively inhibited by the drug (1 or 10 microM) as studied by electron microscopy and by assaying the release of infectious or radiolabeled virus. Immunoelectron microscopy showed that association of viral nucleocapsids with the cytoplasmic surface of glycoprotein-containing Golgi membranes, a prerequisite for virus budding, was unaffected by monensin. In the presence of the drug, the virus glycoproteins assembled into long, tubular structures extending into the lumen of Golgi-derived vacuoles, suggesting that monensin inhibited a terminal step in the assembly of the virus. Intracellular transport and expression of the virus membrane glycoproteins G1 and G2 at the cell surface were not inhibited by monensin as studied by immunocytochemical and radiolabeling techniques. Pulse-chase experiments in the presence of monensin showed that intracellular G1 acquired only partially endo-H-resistant glycans. The sialylation of G1 appearing on the cell surface in the presence of the drug was decreased, whereas sialylation of G2 apparently was inhibited to a lesser extent, as shown by external labeling of the cells with the periodate-boro[3H]hydride method. Thus, monensin exerted a differential effect on the terminal glycosylation of G1 and G2. Unlike several membrane and secretory glycoproteins, both G1 and G2 could enter a functional transport pathway in the presence of monensin and become expressed at the cell surface.

We studied the maturation of Uukuniemi virus and the localization of the viral surface glycoproteins and nucleocapsid protein in infected cells by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy with specific antisera prepared in rabbits against the two glycoproteins G1 and G2 and the nucleocapsid protein N. Electron microscopy of thin sections from infected cells showed virus particles maturing at smooth-surfaced membranes close to the nucleus. Localization of the G1/G2 and N proteins by indirect immunofluorescence at different stages after infection showed the antigens to be present throughout the cell interior but concentrated in the juxtanuclear region. The G1/G2 antiserum also appeared to stain the nuclear and plasma membranes. Double staining with tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, which preferentially stains the Golgi complex, and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, which stained the G1/G2 or N proteins, showed that the staining of the juxtanuclear region coincided. Similarly, double staining for thiamine pyrophosphatase, an enzyme activity specific for the Golgi complex, showed the fluorescence and the cytochemical stain to coincide in the juxtanuclear region. Immunoperoxidase electron microscopy of cells permeabilized with saponin revealed that the viral glycoproteins were present in the rough endoplasmic reticulum and the nuclear and Golgi membranes; the latter was heavily stained. With this method, the N protein was localized to the cytoplasm, especially around smooth-surfaced vesicles in the Golgi region. Taken together, the results indicate that Uukuniemi virus and its structural proteins accumulate in the Golgi complex, supporting the idea that this compartment rather than the plasma membrane is the site of virus maturation. This raises the interesting possibility that deficient transport of the glycoproteins to the plasma membrane and hence their accumulation in the Golgi complex determines the site of virus maturation.

Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 4 is a t-SNARE that, according to previous overexpression studies, is predominantly localized at the plasma membrane. In the present study endogenous syntaxin 4 was found in intracellular vesicular structures in addition to regions of the plasma membrane. In these vesicular structures syntaxin 4 colocalized with rab11, a marker of recycling endosomes. Furthermore, syntaxin 4 colocalized with actin at the dynamic regions of the plasma membrane. Treatment with N-ethylmaleimide, the membrane transport inhibitor, caused an increased accumulation of syntaxin 4/rab11 positive vesicles in actin filament-like structures. Finally, purified recombinant syntaxin 4 but not syntaxin 2 or 3 cosedimented with actin filaments in vitro, suggesting direct interaction between these two proteins. Taken together, these data suggest that syntaxin 4 regulates secretion at the actin-rich areas of the plasma membrane and may be recycled through rab11 positive intracellular membranes.

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [ 3 H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [ 3 H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man) 3 (GlcNAc) 2 peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N -acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N -acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [ 3 H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [ 35 S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Publisher Summary This chapter discusses the use of temperature experiments in studies of protein transport in the exocytic pathway with emphasis on the description of immunocytochemical techniques used in protein detection in light and electron microscropy. The availability of well-characterized soluble and membrane-bound markers made it possible to discover thermosensitive steps in the endocytic pathway. Studies of virus glycoprotein transport have initiated the analysis of the function of distinct intracellular sites in the biosynthetic pathway at which protein movement is arrested at reduced temperatures. Temperature experiments with cultured cells grown on plastic or on glass coverslips require simple equipment. Incubation is most conveniently carried out in water baths adjusted to the appropriate temperature. Bath temperature can be easily monitored with a thermometer punched through the Styrofoam cover, and adjusted, if necessary, by adding a few chucks of ice. At 15°C proteins accumulate in a compartment between the endoplasmic reticulum (ER) and the Golgi complex, whereas at 20°C accumulation occurs at the level of a late-Golgi compartment. The reversible temperature-induced transport blocks provide valuable tools for the morphological and biochemical analysis of membrane traffic. Distinct threshold temperatures exist for different steps in transport pathways, but temperature reduction can also be used to slow down events among different sites and to increase resolution.

The effect of reduced temperature on synchronized transport of SFV membrane proteins from the ER via the Golgi complex to the surface of BHK-21 cells revealed two membrane compartments where transport could be arrested. At 15 degrees C the proteins could leave the ER but failed to enter the Golgi cisternae and accumulated in pre-Golgi vacuolar elements. At 20 degrees C the proteins passed through Golgi stacks but accumulated in trans-Golgi cisternae, vacuoles, and vesicular elements because of a block affecting a distal stage in transport. Both blocks were reversible, allowing study of the synchronous passage of viral membrane proteins through the Golgi complex at high resolution by immunolabeling in electron microscopy. We propose that membrane proteins enter the Golgi stack via tubular extensions of the pre-Golgi vacuolar elements which generate the Golgi cisternae. The proteins pass across the Golgi apparatus following cisternal progression and enter the post-Golgi vacuolar elements to be routed to the cell surface.

Bunyaviruses are enveloped arthropod-borne viruses containing a tripartite single-stranded RNA genome of negative polarity. We have studied the strategy of gene expression of one member of this large family, namely Uukuniemi virus, the prototype of the genus Uukuvirus. Three mRNA species, which do not bind to oligo(dT)-cellulose, are found associated with polysomes of infected cells. Sucrose gradient and gel analyses, in vitro translation and molecular cloning have shown that: (i) The M virion RNA (vRNA) segment is transcribed into an mRNA of roughly the same size (Mr 1.1 × 106) and encodes a precursor (p110) to the two glycoproteins G1 and G2. (ii) Sequence analysis of the 3′ end of the M vRNA, which is complementary to the 5′ end of the M mRNA, indicates an open reading frame starting from an AUG codon 18 nucleotides from the end of the mRNA. (iii) The S vRNA segment (Mr 0.5 × 10 ) is transcribed into a full-length plus-strand RNA (antigenome) and two small mRNA species (Mr about 0.3 × 10°) that encode a nonstructural (NSS) protein (Mr 30,000) and the nucleocapsid (N) protein (Mr 25,000). Whether the two mRNA species contain overlapping nucleotide sequences is not known. By elimination, the L mRNA, transcribed from the L vRNA segment (Mr 2.4 × 106), codes for the L protein (Mr 200,000) the putative RNA polymerase. The results obtained with Uukuniemi virus and other bunyaviruses indicate that the strategy of gene expression of these viruses in some aspects is different from that of other known RNA viruses.

We have studied the transport of the Uukuniemi virus membrane glycoproteins in baby hamster kidney and chick embryo cells by using a temperature-sensitive mutant (ts12). Uukuniemi virus assembles in the Golgi complex, where both glycoproteins G1 and G2 and nucleocapsid protein N accumulate (E. Kuismanen, B. Bång, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984). At the restrictive temperature (39 degrees C), the glycoproteins of ts12 were transported to the Golgi complex as in wild-type, virus-infected cells, whereas the nucleocapsid protein failed to accumulate there. Pulse-chase labeling followed by immunoprecipitation and treatment with endo-beta-N-acetylglucosaminidase H showed that G1 synthesized at 39 degrees C in ts12-infected cells had an altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a lack of terminal glycosylation. The typical Uukuniemi virus-induced vacuolization and expansion of the Golgi complex could be seen also in ts12-infected cells at 39 degrees C, although no virus particles were formed. This suggests that the morphological changes were induced by the Uukuniemi virus glycoproteins. In wild-type virus- or ts12-infected cells, G1 and G2 could not be chased out from the Golgi complex even after 6 h of treatment with cycloheximide. The glycoproteins were thus retained in the Golgi even under conditions when no virus maturation took place and when nucleocapsids did not accumulate in the Golgi region. Accordingly, the glycoproteins of Uukuniemi virus were found to have properties resembling those of Golgi-specific proteins. This virus model system may be useful in studying the synthesis and transport of membrane proteins that are transported to and retained in the Golgi.