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H Garoff

Publications and source records attributed to H Garoff.

At least 73 records · Page 4Linked to original sources
PubMed1987

Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding.

Each subunit of the human transferrin receptor (TR) dimer is inserted into the ER membrane as a transmembrane polypeptide having its N-terminus in the cytoplasm. The transmembrane segment of the molecule serves both as a signal for chain translocation and as a membrane anchor. To study which structural features of this segment are required for its dual function, we have essentially replaced the transmembrane peptide with the C-terminal membrane-spanning segment of two proteins having a separate N-terminal translocation signal and with an artificial uncharged peptide. In each case the mutant TR molecules are efficiently translocated in vitro. In contrast, substitution of the transmembrane peptide of TR with a hydrophilic peptide results in no detectable translocation activity of the mutant TR. This suggests that the hydrophobic character of the transmembrane peptide of TR, rather than its actual amino acid sequence, is important for chain translocation and membrane binding.

Biological Transport↗
PubMed1987

Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells.

We have investigated the role of phosphorylation in the endocytosis of the human transferrin receptor (TR) by replacing its phosphorylation site, Ser24, with Ala through site-directed mutagenesis of the TR cDNA. The TR Ala24 mutant expressed in mouse 3T3 cells was not phosphorylated, even following stimulation of protein kinase C by phorbol ester. However, in spite of this defect the mutant was efficiently endocytosed and recycled back to the plasma membrane with kinetics similar to those of TR and a control mutant TR Ala63. Thus, these results confirm earlier results by Davis et al. (1986, J. Biol. Chem., 261-9034-9041) that Ser24 of human TR is the phosphorylation site for protein kinase C but do not support a role of this modification as a signal for TR endocytosis and recycling.

Animals↗
PubMed1987

Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease.

The protease activities responsible for the cotranslational processing of the Semliki Forest virus structural polyprotein were investigated by using an in vitro transcription-translation system. Three cleavages released the individual chains from the nascent polyprotein in the order capsid, p62, 6K (a nonstructural peptide), and E1. We showed directly that the protease activity responsible for the release of the capsid protein resides in the capsid itself: by progressive truncation of the cDNA used for the SP6 transcription, we showed that a precursor containing as few as 38 residues of the p62 protein left at the C terminus of the capsid was still very efficiently cleaved in vitro. We further tested the possibility that serine-219 of the capsid is involved in autoproteolysis by site-directed in vitro mutagenesis. A change in the sequence Gly-Asp-Ser(219)-Gly, a tetrapeptide conserved among several animal serine proteases, to Gly-Asp-Arg-Ser-Thr was shown to completely abolish in vitro cleavage. This supports the notion that the capsid is a serine protease. The role of the capsid protease in the processing of the 6K junctions was then investigated by translations of a hybrid polyprotein in which the capsid and most of the p62 sequences are replaced by those of the secretory protein lysozyme. The cleavages and concomitant appearance of the 6K peptide occurred efficiently and were shown to require the presence of membranes. This demonstrates that the capsid protease is not required for those cleavages and suggests that a membrane-associated host protease is responsible for the cleavage.

Capsid↗
PubMed1986

The transmembrane segment of the human transferrin receptor functions as a signal peptide.

The human transferrin receptor (TR) is a protein comprising 760 amino acid residues that spans the membrane once with its N terminus towards the cytoplasm. It is synthesized without a cleavable signal peptide. We have tested whether the signal responsible for its membrane insertion is present within its transmembrane peptide using a combined recombinant DNA/in vitro translation approach. The complete TR coding region was first reconstructed from overlapping TR cDNA clones and then engineered into an SP6-based transcription vector. In vitro transcription and subsequent translation in the presence of rough microsomes yielded TR molecules that were glycosylated and correctly inserted into the membrane. Two kinds of experiments demonstrated that the spanning region of the TR polypeptide contained the signal for translocation across the membrane of the rough endoplasmic reticulum. First, we deleted the spanning region of TR and showed that this deletion mutant could not be inserted. Second, we showed that two cytoplasmic proteins (the mouse dihydrofolate reductase and the chimpanzee alpha-globin) could be inserted into the microsomal membrane in the expected orientation when the TR transmembrane segment was added to their N termini. Thus, the spanning peptide was shown to be both necessary and sufficient for chain translocation. Further analyses demonstrated that the translocation event was dependent on the signal recognition particle.

Cell Membrane↗
PubMed1986

Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal for the E1 glycoprotein.

The biosynthesis of the Semliki Forest virus (SFV) structural proteins provides an interesting model system to study the reinitiation of translocation of membrane proteins into the endoplasmic reticulum membrane. The two transmembrane spike proteins, p62 and E1, are derived from a single polypeptide precursor. Once the first protein, p62, has been anchored and its cytoplasmic tail has been synthesized, translocation must be reinitiated to account for the insertion of the E1 protein. We have used deletion mutagenesis of the SFV cDNA to investigate the requirements for this event and map in detail the location of the signal. We have shown by deleting the region encoding the p62 signal and expressing the modified cDNA in COS cells that the p62 protein is not involved in the translocation of the E1 protein. The E1 signal was precisely mapped by progressive truncations of the 6 K peptide (located between p62 and E1 in the SFV polyprotein) and subsequent analysis in cell-free systems. A segment within the last 26 residues of the 6 K peptide was shown to be essential for translocation. This segment was then fused to the N-terminus of the chimpanzee alpha-globin and was shown to be sufficient for translocation. The E1 signal was cleaved efficiently even when attached to the alpha-globin protein. The activity of the signal was found to be SRP dependent in a wheat-germ cell-free system. We conclude that prior attachment of the ribosome to the membrane via the p62 signal peptide is not necessary for E1 translocation and that the reinitiation of translocation is mediated by an independent internal signal likely to be SRP dependent.

Amino Acid Sequence↗
PubMed1986

Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity.

Three mutations of the membrane-binding region of the Semliki Forest virus (SFV) p62 polypeptide (the precursor for virion E3 and E2) have been made by oligonucleotide-directed mutagenesis of a cDNA clone encoding the SFV structural proteins. One of the mutations (A2) substitutes a Glu for an Ala in the middle of the hydrophobic stretch which spans the bilayer. A1 and A3 alter the two basic charged amino acids in the cytoplasmic domain next to the hydrophobic region. The wild-type charge cluster of Arg-Ser-Lys (+2) has been changed to Gly-Ser-Met (0;A3) or to Gly-Ser-Glu (-1;A1). The mutant p62 proteins have been analyzed both in the presence and the absence of E1, the other half of the heterodimer spike complex of SFV. The mutant proteins expressed in COS-7 cells are glycosylated and are of the expected sizes. When co-expressed with E1, all three mutants are cleaved to yield the E2 protein and transported to the surface of COS-7 cells. When expressed in the absence of E1, the mutant p62 proteins remain uncleaved but still reach the cell surface. Once at the cell surface, all three mutants, when co-expressed with E1, can promote low pH-triggered cell-cell fusion. These results show that the three mutant p62/E2 proteins are still membrane associated in a functionally unaltered way.

Amino Acid Sequence↗
PubMed1986

Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding.

The p62/E2 protein of Semliki Forest virus (SFV) is a typical transmembrane glycoprotein, with an amino-terminal lumenal domain, a transmembrane (hydrophobic) domain, and a carboxy-terminal cytoplasmic domain (or tail). Our hypothesis has been that the membrane-binding polypeptide region (membrane anchor) of this protein consists of both the transmembrane domain and the adjacent positively charged peptide, Arg-Ser-Lys, which is part of the cytoplasmic domain. We have investigated three anchor mutants of the p62 protein with respect to both their disposition and their stability in cell membranes. The construction of the three mutants has been described (Cutler, D.F., and H. Garoff, J. Cell Biol., 102:889-901). They are as follows: A1, changing the basic charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Glu(-1); A2, replacing an Ala in the middle of the hydrophobic stretch with a Glu; A3, changing the charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Met(0). All three mutants retain the transmembrane configuration of the wild-type p62. In a cell homogenate they have a cytoplasmic domain that is accessible to protease. In living cells an anti-peptide antibody specific for the cytoplasmic tail of p62 reacts with the tails of both wild-type and mutant p62s following its introduction into the cytoplasm. All three mutant proteins have Triton X-114 binding properties similar to the wild-type p62. However, when the membranes of cells expressing the three mutants or the wild-type p62 protein are washed with sodium carbonate, pH 11.5, three to four times as much mutant protein as wild-type p62 is released from the membranes. Thus the stability in cell membranes of the three mutant p62 proteins is significantly reduced.

Amino Acid Sequence↗
PubMed1986

Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells.

Expression of the Semliki Forest virus p62/E2 protein was studied in the polarized epithelial cell line Madin-Darby canine kidney (MDCK). After infection this transmembrane protein, together with the other spike subunit E1, accumulates at the basolateral surface of MDCK cells (Fuller, S. D., C.-H. von Bonsdorff, and K. Simons, 1985, EMBO (Eur. Mol. Biol. Organ.) J., 4:2475-2485). The cDNAs encoding truncated forms of the protein were used to stably transform MDCK cells to examine the role of subunit oligomerization (E1-E2) and the cytoplasmic domain of p62/E2 in directed transport to the basolateral surface. The biochemical characteristics and polarity of the expressed proteins were studied using cell monolayers grown on nitrocellulose filters. A wild-type form of p62/E2, in the absence of E1, and two forms having either 15 or 3 of the wild-type 31-amino acid carboxyl cytoplasmic domain were all localized to the basolateral surface. These results indicate that the cytoplasmic domain of E2 does not contain the information essential for directed transport to the plasma membrane, and imply that this information resides in either the lumenal and/or membrane-spanning segments of this transmembrane protein.

Animals↗
PubMed1985

Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK.

Secretion of a foreign protein--chicken oviduct lysozyme--and of endogenous proteins was studied in the polarized epithelial Madin-Darby Canine Kidney (MDCK) cell line. Cell clones that secrete enzymatically active chicken lysozyme were generated by transforming the cells with lysozyme cDNA inserted in a SV40-pBR322 recombinant vector and a dominant selectable marker gene. The kinetics and polarity of lysozyme secretion from one transformed cell clone were studied using cell monolayers grown on nitrocellulose filters. Lysozyme was secreted into the apical and the basolateral medium, demonstrating the existence of direct transport pathways to each cell surface. Control experiments excluded the effects of monolayer leakiness, reabsorption, transepithelial transport, and depolarization. In contrast, the secretion of a set of endogenous proteins of MW 30-40 kd was found to be strictly apical showing that polarized secretion also occurs in this cell line. The latter group of proteins appear to be generated from larger precursor molecules by intracellular cleavage.

Animals↗
PubMed1985

Secretion of Semliki Forest virus membrane glycoprotein E1 from Bacillus subtilis.

The gene coding for the Semliki Forest virus (SFV) membrane protein E1 was joined to a secretion vector containing the promoter and signal sequence regions of the alpha-amylase gene from Bacillus amyloliquefaciens. To facilitate secretion, the regions coding for the N-terminal signal peptide (the 6K protein) and the C-terminal hydrophobic transmembrane domain of the E1 gene were deleted. After transformation into B. Subtilis, E1 was shown by immunoblotting to be expressed at a low level (about 0.5-1 mg/1). Contrary to what was expected, most of the E1 remained cell-associated. Deletion of a residual 7 C-terminal amino acids from the 6K region neither increased the level of expression nor significantly improved the secretion. Immunofluorescence microscopy of protoplasts prepared from B. subtilis cells expressing E1 suggested that the cell-associated E1 was located at the outer surface of the bacterial membrane. Addition of protease inhibitors to the culture medium somewhat increased the amount of extracellular E1, suggesting that proteolytic degradation of the foreign gene product may be one reason for the low level of expression. This conclusion was also supported by experiments carried out in Bacillus minicells, which indicated that the expression of the E1 gene in the absence of synthesis of bacterial proteases was about the same as that of alpha-amylase expressed from the cloned gene using the same promoter and signal sequence.

Bacillus subtilis↗
PubMed1984

Cell surface expression of fusogenic vesicular stomatitis virus G protein from cloned cDNA.

Vesicular stomatitis virus (VSV) enters the host cell by the receptor-mediated endocytotic pathway. This brings the virus particle into acidic vesicles inside the cell where infection occurs through a fusion event between the viral and the host vesicle membrane. In this work we have shown that the VSV glycoprotein (G) carries the fusion activity of this virus. The G protein was expressed on the surface of baby hamster kidney 21 cells from cloned cDNA which had been engineered into an expression vector and introduced into cell nuclei with the aid of a glass microneedle. A short (60 s) treatment with acid (pH less than or equal to 6.0) medium induced fusion of cells having G protein on their surface. For efficient G protein expression and cell-cell fusion we had to trim the 5' end of the G cDNA and to use as promoter the long terminal repeat of the mouse Moloney sarcoma virus.

Animals↗
PubMed1983

Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein.

A complementary (cDNA) molecule encoding the structural proteins of Semliki Forest virus (SFV) has been inserted into a Simian virus 40-derived eucaryotic expression vector lacking introns. Introduction of the recombinant DNA into nuclei of baby hamster kidney cells results in the synthesis of authentic SFV membrane glycoproteins E1 and E2. The glycoproteins are both transported to the cell surface and induce cell-cell fusion after a brief treatment of the cells with low pH medium. The pH dependence of the fusion reaction was the same as that induced by virus particles (White, J., J. Kartenbeck, and A. Helenius, 1980, J. Cell Biol., 89:674-679). Transfection of cells with another recombinant DNA molecule in which the SFV cDNA is engineered into the same expression vector including an intron has been shown before to result in the expression of only the E2 protein on the cell surface, whereas the E1 protein is trapped in the rough endoplasmic reticulum (Kondor-Koch, C., H. Riedel, K. Söderberg, and H. Garoff, 1982, Proc. Natl. Acad. Sci. USA, 79:4525-4529). Such cells do not exhibit pH-dependent polykaryon formation, suggesting that the E1 protein is necessary for fusion activity. Immunoblotting experiments show that the RER-trapped E1 protein expressed from the DNA construction with an intron has a smaller apparent molecular weight than authentic E1, and that is has lost its amphipathic characteristics.

Animals↗
PubMed1983

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.

Amino Acid Sequence↗
PubMed1982

A procedure to verify an amino acid sequence which has been derived from a nucleotide sequence: application to the 26S RNA of Semliki Forest virus.

We describe a peptide sequencing procedure which can be used to verify an amino acid sequence which is derived from a nucleotide sequence. One first labels the protein with a 3H- and a 14C-labelled amino acid and then cleaves the protein into a set of peptides using a cleavage reaction specific for a particular amino acid residue. Finally one performs Edman degradations on the whole mixture of peptides. The released amino acids reflect the combined aminoterminal amino acid sequences of all the peptides that have been formed by the cleavage reaction. The data can therefore be used to check a deduced sequence simultaneously at several regions of the polypeptide chain. We have applied this sequencing procedure to verify the amino acid sequence deduced from the 26S RNA of Semliki Forest virus.

Amino Acid Sequence↗