N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.

Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.     

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.     

VAT-1: an abundant membrane protein from Torpedo cholinergic synaptic vesicles

Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays. The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions. An antibody raised against a fusion protein synthesized in E. coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles. Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein. The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals. These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles.     

Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation- specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.     

A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

The Euglena precursor to the small subunit of ribulose-15-bisphosphate carboxylase/oxygenase (pSSU) is a polyprotein. To determine the transport route from cytoplasm to chloroplast, Euglena was pulse labeled with 35S-sulfate and the organelles were separated on sucrose gradients. After a pulse, pSSU was found in the endoplasmic reticulum (ER) and Golgi apparatus. During a chase, ER-and Golgi-localized pSSU decreased concomitant with the appearance of SSU in chloroplasts. SSU was not found in pSSU-containing ER and Golgi fractions. Na2CO3 did not remove pSSU from ER or Golgi membranes, indicating that it was an integral membrane protein. pSSU was inserted in vitro into canine microsomes, and Na2CO3 did not remove pSSU from the microsomal membrane. The in vivo and in vitro experiments show that Euglena pSSU is inserted into the ER membrane and transported as an integral membrane protein to the Golgi apparatus before chloroplast import and polyprotein processing.     

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.     

Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor

An N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) has recently been purified on the basis of its ability to restore transport to NEM-inactivated Golgi membranes in a cell-free transport system. NSF is a peripheral membrane protein required for the fusion of transport vesicles. We now report the existence of two novel components that together bind NSF to Golgi membranes in a saturable manner. These components were detected by examining the requirements for reassociation of purified NSF with Golgi membranes in vitro. One component is an integral membrane receptor that is heat sensitive, but resistant to Na2CO3 extraction and to all proteases tested. The second component is a cytosolic factor that is sensitive to both proteases and heat. This soluble NSF attachment protein (SNAP) is largely resistant to NEM and is further distinguished from NSF by chromatography. SNAP appears to act stoichiometrically in promoting a high-affinity interaction between NSF and the membrane receptor. Because NSF promotes vesicle fusion, it seems likely that these two new factors that allow NSF to bind to the membrane are also part of the fusion machinery.     

DSCR2, a Down syndrome critical region protein, is localized to the endoplasmic reticulum of mammalian cells

We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2) in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER) of cells. We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM) helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.     

Expression of Semliki Forest virus proteins from cloned complementary DNA. II. The membrane-spanning glycoprotein E2 is transported to the cell surface without its normal cytoplasmic domain

The E2 protein (422 amino acid residues long) of Semliki Forest virus is a spanning membrane protein which is made in the rough endoplasmic reticulum of the infected cell and transported to the cell surface. The cytoplasmic domain of this protein comprises 31 amino acid residues. We introduced deletions of various sizes into the gene region encoding this part of the protein molecule and analyzed the transport behavior of the mutant proteins. The deletions were made using exonuclease digestions of cloned cDNA encoding the E2 protein. When the mutated DNA molecules, engineered into an expression vector, were introduced into nuclei of baby hamster kidney 21 cells, membrane proteins with cytoplasmic deletions were expressed and routed to the cell surface in the same way as the wild-type protein. This suggests that the cytoplasmic domain of the E2 protein does not carry information that is needed for its transport from the rough endoplasmic reticulum to the cell surface.     

Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein

We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.     

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.     

The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane.

We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre-sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane.     

Evidence for the localization of the nuclear-coded 22-kDa heat-shock protein in a subfraction of thylakoid membranes.

The precursor to the nuclear-coded 22-kDa heat-shock protein of chloroplasts (HSP 22) has been transported into isolated intact chloroplasts from heat-shocked plants. The localization of the mature protein in the chloroplast membrane was investigated. We have shown that the processed HSP 22 of pea was not bound to envelopes and found predominantly in thylakoid membranes. The binding of HSP 22 was stable in the presence of high salt concentrations. Solubilization of thylakoid membranes with Triton X-100 and phase partitioning with Triton X-114 indicate an intrinsic localization of HSP 22 or, alternatively, a non-covalent association with integral membrane protein(s). After fractionation into grana and stroma lamellae, HSP 22 was found mostly in the grana-membrane subfraction.     

Integral membrane protein located in the apical complex of Plasmodium falciparum.

We describe the cloning of a novel antigen of Plasmodium falciparum which contains a hydrophobic domain typical of an integral membrane protein. This antigen is designated apical membrane antigen 1 because it appears to be located in the apical complex. Apical membrane antigen 1 appears to be transported to the merozoite surface near the time of schizont rupture.     

Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6

PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.     

Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide.

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals, we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately 100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s function to direct portions of the molecule across the membrane and are able to perform this function even when positioned in an internal protein domain.     

A fusion-defective mutant of the vesicular stomatitis virus glycoprotein.

We have recently described an assay in which a temperature-sensitive mutant of vesicular stomatitis virus (VSV; mutant tsO45), encoding a glycoprotein that is not transported to the cell surface, can be rescued by expression of wild-type VSV glycoproteins from cDNA (M. Whitt, L. Chong, and J. Rose, J. Virol. 63:3569-3578, 1989). Here we examined the ability of mutant G proteins to rescue tsO45. We found that one mutant protein (QN-1) having an additional N-linked oligosaccharide at amino acid 117 in the extracellular domain was incorporated into VSV virions but that the virions containing this glycoprotein were not infectious. Further analysis showed that virus particles containing the mutant protein would bind to cells and were endocytosed with kinetics identical to those of virions rescued with wild-type G protein. We also found that QN-1 lacked the normal membrane fusion activity characteristic of wild-type G protein. The absence of fusion activity appears to explain lack of particle infectivity. The proximity of the new glycosylation site to a sequence of 19 uncharged amino acids (residues 118 to 136) that is conserved in the glycoproteins of the two VSV serotypes suggests that this region may be involved in membrane fusion. The mutant glycoprotein also interferes strongly with rescue of virus by wild-type G protein. The strong interference may result from formation of heterotrimers that lack fusion activity.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.