Sufficient Hydrophobicity Research Articles

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of gamma-secretase are discussed.

Homogeneous reversed-phase agarose thermogels for electrochromatography

A method for the derivatization of agarose by covalent attachment of hydrophobic ligands for reversed-phase (RP) chromatographic separation and ionic groups for generation of electroosmosis under electrochromatographic conditions in the capillaries or microfluidic channels filled with the thermogel of this agarose derivative is described. The product renders a capability of reversible thermogelation. The thermogels formed provide sufficient hydrophobicity and electroosmosis for the separations of the analytes under RP mobile-phase conditions and electric field applied. The gels may be used repeatedly without loss of resolution. They are thermally replaceable and UV transparent (providing possibility in column/in-gel detection), require no covalent attachment to the capillary inner wall (or microchip channel), and are suitable for isocratic or gradient operation in the aqueous–organic mobile phases.

GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery

GALA is a 30 amino acid synthetic peptide with a glutamic acid-alanine-leucine-alanine (EALA) repeat that also contains a histidine and tryptophan residue as spectroscopic probes. It was designed to explore how viral fusion protein sequences interact with membranes. The sequence selected was long enough to span a bilayer in the α-helix, the glutamic acids (Glu) were selected to provide a pH-dependent negatively charged side-chain and the EALA repeat was adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when the peptide was in an α-helix. GALA converts from a random coil to an amphipathic α-helix when the pH is reduced from 7.0 to 5.0. At neutral pH, GALA is water soluble while at acid pH, GALA binds to bilayer membranes. The nature of the association and the type of peptide–peptide interactions in the membrane depend upon the physico-chemical properties of the bilayer such as the acyl chain composition of the phospholipids and the presence of cholesterol. Neutral and negatively charged bilayers composed of saturated phospholipids of 14–16 acyl chain length are solubilized into peptide–lipid discs by GALA. GALA can induce fusion between small unilamellar vesicles (SUV) composed of unsaturated phospholipids. Most importantly GALA forms a transmembrane peptide pore comprised of approximately 10 GALA α-helical monomers that are arrayed in an α-helix perpendicular to the plane of the membrane. Membrane leakage from neutral or negatively charged vesicles at pH 5.0 can be adequately explained by a mathematical model assuming that GALA becomes incorporated into the vesicle bilayer and aggregates to form a transbilayer pore consisting of 10 (±2) peptides. The lipid compositions of model bilayer have important effects on the GALA transbilayer insertion mechanism and peptide orientation. Insertion of the pore into the membrane dramatically accelerates transmembrane phospholipid flip-flop. Cationic peptides designed based upon GALA but containing a lysine-alanine-leucine-alanine (KALA) motif can interact with nucleic acids and perturb biomembranes. The pH-controlled membrane permealization induced by GALA and related peptides serve as a paradigm for the design of environmentally responsive peptidic delivery vehicles for drugs and genes.

Invesgations [correction investigations] on the influence of halide substituents on the estrogen receptor interaction of 2, 4, 5-tris(4-hydroxyphenyl)imidazoles.

Previously, we reported on the synthesis and estrogen receptor (ER) interaction of imidazoles, which had to be 1-alkyl-4, 5-bis(2-halo-4-hydroxyphenyl) substituted for a high relative binding affinity (RBA >1 %). This led to the assumption that a shielding of the polar heterocyclic system is a prerequisite for ER binding. In continuation of this study we synthesized 2, 4, 5-tris(4-hydroxyphenyl)imidazoles with Cl-or F-atoms in the ortho-positions of the aromatic rings and evaluated whether they mediate sufficient hydrophobicity for ER interaction. 2-(2, 6-Dichloro-3/4-hydroxyphenyl)-4, 5-bis(2-halo-4-hydroxyphenyl)imidazoles were synthesized by reaction of the respective methoxy-substituted benzil with either the 2, 6-dichloro-4-methoxy-or the 2, 6-dichloro-3-methoxybenzaldehyde in ammonium acetate solution. The required ether cleavage was performed subsequently with BBr(3). In the competition experiment with [(3)H]estradiol the imidazoles with the a C2-standing (2, 6-dichloro-4-hydroxyphenyl) ring showed an RBA >0.02 %, but did not activate the luciferase gene in estrogen receptor positive MCF-7-2a breast cancer cells stably transfected with the plasmid ERE(wtc)luc. In the test for antagonistic potency only the 2-(2, 6-dichloro-4-hydroxyphenyl)-4, 5-bis(4-hydroxyphenyl)imidazole 3 antagonized the effects of 1 nM estradiol slightly. From these data, it can be concluded that a C2-standing 2, 6-dichloro-4-hydroxyphenyl ring is not appropriate to optimize the ER interaction of 4, 5-(4-hydroxyphenyl)imidazoles.

03/01771 Apparatus for carbonization production of bamboo charcoal and bamboo pyroligneous vinegar liquid: Nishimura, T. Jpn. Kokai Tokkyo Koho JP 2002 332,488 (Cl. C10B53/02), 22 Nov 2002, Appl. 2001/141,258. (In Japanese)

In this study, bamboo fiber filled polypropylene composites (BPCs) were prepared using bamboo and coconut shell charcoal as reinforcing fillers for enhancing the properties of composites. Bamboo and coconut shell biomass were carbonized at different carbonization temperatures (300, 400, 600, and 900OC); 1 h soaking time and at 5OC−min heating rates. The effect of carbonization temperature and charcoal content (0.5%, 1.0% and 1.5%) on mechanical, thermal and water absorption properties of BPCs has been investigated. The experimental results show that charcoal which is obtained at lower pyrolysis temperature (300OC and 400OC) improves the strength properties of BPCs, whereas, strength properties remains unaltered when charcoal prepared at elevated temperatures (600OC and 900OC) are added to BPCs. Compared to coconut shell charcoal (CSC) better mechanical properties were obtained through bamboo charcoal (BC). This has been attributed to flakes like appearance of BC, having pores present on their surfaces. The study also shows that addition of charcoal provides thermal stability to BPCs, particularly via charcoal prepared at elevated temperature (900OC). The optimum temperature for achieving sufficient hydrophobicity in BPCs is found to be 300OC and 600OC for BC and CSC, respectively.

The role of the abundant phenylalanines in the mode of action of the antimicrobial peptide clavanin

Clavanin A is a special antimicrobial peptide that acts at the level of the membrane via a pH-dependent mechanism. At neutral pH, clavanin disrupts biological and model membranes in a nonspecific manner, causing efflux of large molecules. At mildly acidic conditions, however, the peptide efficiently kills bacteria by permeabilizing their membrane most likely by interacting with proteins involved in proton translocation [Biochemistry 41 (2002) 7529]. Clavanin A is unusually rich in phenylalanines with 5 out of 23 residues, which suggests that these residues are functionally important. A set of mutants, in which all Phe residues are replaced by either Ile, Leu, Trp, or Tyr was used to investigate the role of these amino acids. The antimicrobial activities of the different peptides both at neutral and low pH show that the presence of phenylalanine is not essential nor optimal, as the Trp, Leu, and Ile mutant are equally or more active than the wild-type component. In general, at neutral pH, the biological activities correlate well with the peptides' ability to interact with membrane lipids. Correspondingly, the permeabilization efficiencies of biological and model membranes of the various derivatives were found to be closely related to their ability to adopt α-helical structures, and follows the order 5L>5W>5I>5Y>wild type. The results suggest an important role for the Phe residues, in providing the peptide in a balanced manner with sufficient hydrophobicity, and therewith membrane affinity, as well as conformational flexibility.

Ion-pair extraction behavior of divalent metal cations using neutral di-Schiff base ligands derived from 1,2-cyclohexanediamine and o-phenylenediamine

Di-Schiff base ligand, N, N'-bis(2-pyridylmethylidene)- trans-1,2-diiminocyclohexane ( trans-BPIC), having sufficient hydrophobicity acts as neutral bidentate ligand in ion-pair extraction of divalent metal cations into nitrobenzene with picrate anion. In present study, the effect of steric restriction by chemical structure around imine-N donor atoms in trans-BPIC analogs on their complexation with divalent metal cations in ion-pair extraction was investigated by using N, N'-bis(2-pyridylmethylidene)- cis-1,2-diiminocyclohexane ( cis-BPIC) and N, N'-bis(2-pyridylmethylidene)- o-diiminobenezene (BPIB). The former was used to observe the effect by geometrical restriction and the latter was by conjugate restriction. In BPIB–NaPic system, the higher extractability was obtained than those in cis- and trans-BPIC systems, and this result seems to be led by the increase of steric distortion originated from conformational restriction. Namely, it is considered that the extractability can be controlled by steric restriction on the complexation.

Changes in the Lipid Composition of Saccharomyces cerevisiae Race Capensis (G-1) During Alcoholic Fermentation and Flor Film Formation

The cellular lipid composition of one flor-forming strain of Saccharomyces cerevisiae during fermentation and the subsequent period of film formation with different oxygen levels was studied. Irrespective of fermentation conditions, only those yeasts which came into contact with oxygen after fermentation formed a flor film. After the fermentation, these yeasts entered an adaptation phase in which the percentage of oleic acid increased considerably at the expense of other long-chain fatty acids. Their phospholipid contents remained high, as well as the unsaturation index of their fatty acids and the ergosterol/phospholipids ratio was maintained below 1. These changes allowed an increased viability of yeasts in the wine of up to 80% and the acquisition of sufficient hydrophobicity and floatability to reach the surface and form flor film.

Effects of activated carbon surface chemistry and pore structure on the adsorption of organic contaminants from aqueous solution

The objective of this research was to develop activated carbon selection criteria that assure the effective removal of trace organic contaminants from aqueous solution and to base the selection criteria on physical and chemical adsorbent characteristics. To systematically evaluate pore structure and surface chemistry effects, a matrix of activated carbon fibers (ACFs) with three activation levels and four surface chemistry levels was prepared and characterized. In addition, three granular activated carbons (GACs) were studied. Two common drinking water contaminants, relatively polar methyl tertiary-butyl ether (MTBE) and relatively nonpolar trichloroethene (TCE), served as adsorbate probes. TCE adsorbed primarily in micropores in the 7–10 Å width range while MTBE adsorbed primarily in micropores in the 8–11 Å width range. These results suggest that effective adsorbents should exhibit a large volume of micropores with widths that are about 1.3 to 1.8 times larger than the kinetic diameter of the target adsorbate. Hydrophobic adsorbents more effectively removed both TCE and MTBE from aqueous solution than hydrophilic adsorbents, a result that was explained by enhanced water adsorption on hydrophilic surfaces. To assure sufficient adsorbent hydrophobicity, the oxygen and nitrogen contents of an activated carbon should therefore sum to no more than about 2 to 3 mmol/g.

Obtenção de oligossacarídeos N-ligados às glicoproteínas dos líquens Sticta tomentosa e Sticta damaecornis

As glicoproteínas dos líquens Sticta tomentosa e Sticta damaecornis foram extraídas utilizando-se tampão específico e fracionadas pela adição crescente de sulfato de amônio. Dentre os diferentes cortes de saturação obtidos, as frações 30-80% de ambos os líquens foram eleitas objeto de estudo desta pesquisa. Métodos químicos e enzimáticos foram aplicados para a obtenção de oligossacarídeos N-ligados, que em seguida, foram derivatizados resultando em tirosinamida-oligossacarídeos. Após inserção do grupo cromóforo nas estruturas oligossacarídicas, os mesmos foram purificados por HPLC com detecção em 280 nm. Em relação ao líquen Sticta tomentosa, os cromatogramas revelaram a presença de dois picos com tempos de retenção de aproximadamente 11 e 18 minutos sugerindo a presença de dois diferentes oligossacarídeos N-ligados. O líquen Sticta damaecornis, seguindo as mesmas condições de purificação, apresentou em cromatografia quatro picos distintos com tempos de 11, 13, 18,2 e 18,4 minutos, respectivamente, sugerindo por sua vez a presença de quatro oligossacarídeos N-ligados diferentes.

Enrichment of G-protein palmitoyltransferase activity in low density membranes: in vitro reconstitution of Galphai to these domains requires palmitoyltransferase activity.

Many signaling proteins are targeted to low density, sphingomyelin- and cholesterol-enriched membranes, also called lipid rafts. These domains organize receptor-mediated signaling events at the plasma membrane. Fatty acylation is one mechanism for targeting proteins to rafts. It was therefore of interest to determine if protein palmitoyltransferase activity is also present in these domains. In this study, protein palmitoyltransferase activity, assayed using G-protein alpha subunits as a substrate, was found to be highly enriched in low density membranes derived from cells that express caveolin as well as those that do not. Depletion of cellular cholesterol with the drug methyl-beta-cyclodextrin resulted in inhibition of palmitoyltransferase activity and a redistribution of the remaining activity to membranes of higher density. This effect was reversed by adding cholesterol to cyclodextrin-treated cells. When reconstituted into cell membranes, the population of purified recombinant G(alphai) that was palmitoylated was highly enriched in the low density membrane fractions, whereas the bulk unmodified G(alphai)-protein was largely excluded. This effect required palmitoyltransferase activity and was abolished if the palmitoylated cysteine was mutated. Thus, palmitoyltransferase facilitates the enrichment of fatty acylated signaling molecules in plasma membrane subdomains.

Quenching of bacteriochlorophyll fluorescence in chlorosomes from Chloroflexus aurantiacus by exogenous quinones.

The quenching of bacteriochlorophyll (BChl) c fluorescence in chlorosomes isolated from Chloroflexus aurantiacus was examined by the addition of various benzoquinones, naphthoquinones (NQ), and anthraquinones (AQ). Many quinones showed strong quenching in the micromolar or submicromolar range. The number of quinone molecules bound to the chlorosomes was estimated to be as small as one quinone molecule per 50 BChl c molecules. Quinones which exhibit a high quenching effect have sufficient hydrophobicity and one or more hydroxyl groups in the alpha positions of NQ and AQ. Chlorobiumquinone has been suggested to be essential for the endogenous quenching of chlorosome fluorescence in Chlorobium tepidum under oxic conditions. We suggest that the quenching effect of chlorobiumquinone in chlorosomes from Chl. tepidum is related to the 1'-oxo group neighboring the dicarbonyl group.

Identification and structure of the Marek's disease virus serotype 2 glycoprotein M gene: comparison with glycoprotein M genes of Herpesviridae family.

We determined the nucleotide sequence of a portion of BamHI-C fragment of Marek's disease virus serotype 2 (MDV2) strain HPRS24 which was suspected to contain the homologue of the herpes simplex virus type 1 (HSV-1) gene UL10, encoding glycoprotein M (gM). An open reading frame whose translation product exhibited significant similarities to HSV-1 gM protein and respective proteins of other herpesviruses of 37.5% and 45.5% to 31.8%, respectively, was identified. A number of distinct transcriptional consensus sequences were found upstream of the first putative start codon of MDV2 UL10 protein. In transcriptional analysis, the gene was transcribed into an 1.5 kb RNA. The primary translation product comprises 424 amino acids with a predicted molecular weight of 46.9 kDa. The predicted MDV2 UL10 protein contains eight hydrophobic domains with sufficient length and hydrophobicity to span the lipid bilayer conserved in the genomes of all herpesviruses which have been sequenced so far. In the region located between the first and second hydrophobic domains, two potential N-linked glycosylation sites were presented. Interestingly, highly charged residues were abundantly possessed in the carboxy-terminal part of the MDV2 UL10 protein. By comparison of the amino acid sequence of the MDV2 UL10 gene with the homologues from other herpesviruses, the data might contribute for further evidence of the evolution of herpesviruses from a common progenitor and an ancient example of MDV2 belonging to the Alphaherpesvirinae subfamily. In addition, the existence of corresponding genes in human, mammalian, and avian herpesvirus genomes, suggests indirectly an important role for gM in the natural life cycle of the virus.

Linear poly(ethylene oxide)-based polyurethane hydrogels: polyurethane-ureas and polyurethane-amides.

Over the last 30 years, water-swellable and water-insoluble hydrogels have been extensively investigated and developed, leading to a large family of materials which have found uses in a wide range of biomedical applications. While hydrogels usually present a crosslinked structure, linear polyurethane-ureas (PUUs) based on poly(ethylene oxide) have been shown to be able to absorb and swell with aqueous media without dissolving. This behavior is due to the phase separated domain morphology, where hydrogen bonded urethane/urea hard segment domains are dispersed in a PEO soft segment domain. This work investigates the possibility of obtaining linear poly(ethylene oxide)-based polyurethane-amide (PUA) hydrogels using two amide diols as chain extenders, a mono amide diol (AD) and a diamide diol (DD), and a dicarboxylic acid (maleic acid, MA). Poly(ethylene oxide) based PUAs were obtained using a "one-shot" bulk polymerization technique. The chemicophysical characterization and water-solubility tests showed that these materials, while having molecular weights similar to the PUUs, do not possess sufficient phase separation, hydrogen bonding and hydrophobicity of the hard segment domains to exhibit hydrogel behavior. Crosslinked PUAs using maleic acid as chain extender show interesting hydrogel properties.

Polypeptides for controlled release applications: Synthesis and preliminary characterization and release studies

The objective of this study is to evaluate the feasibility of synthesizing biodegradable polypeptide polymers of sufficient hydrophobicity so that release of solute is diffusion controlled. The benefit of such a diffusion-controlled, biodegradable polypeptide system is that the main advantages of biodegradable (avoiding the retrieval of the implanted device) and non-degradable polymers (greater structural integrity) are both exploited. In this study, one sequential polypeptide and two random co-polypeptides with different hydrophobicities were synthesized using active ester intermediates. The polymers were characterized with regard to their chemical composition, molecular weight and hydrophobicity. The in vitro release of a model steroid, 17 α-ethynylestradiol, from compressed disks of these polymers followed a square root of time dependence indicating a diffusion controlled release mechanism. The drug release rates could be varied over several orders of magnitude and covered the therapeutic range for an implantable device delivering the steroid drug on the order of 10 mg per day. The results of these studies indicate that polypeptide co-polymers can be made with sufficient hydrophobicity to provide release rates in the therapeutic range for parenteral drug delivery.

Interactions among Subunits of the Oligosaccharyltransferase Complex

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

97/01172 Effect of properties of Chinese coals on activated carbon characteristics

The objective of this research was to develop activated carbon selection criteria that assure the effective removal of trace organic contaminants from aqueous solution and to base the selection criteria on physical and chemical adsorbent characteristics. To systematically evaluate pore structure and surface chemistry effects, a matrix of activated carbon fibers (ACFs) with three activation levels and four surface chemistry levels was prepared and characterized. In addition, three granular activated carbons (GACs) were studied. Two common drinking water contaminants, relatively polar methyl tertiary-butyl ether (MTBE) and relatively nonpolar trichloroethene (TCE), served as adsorbate probes. TCE adsorbed primarily in micropores in the 7–10 Å width range while MTBE adsorbed primarily in micropores in the 8–11 Å width range. These results suggest that effective adsorbents should exhibit a large volume of micropores with widths that are about 1.3 to 1.8 times larger than the kinetic diameter of the target adsorbate. Hydrophobic adsorbents more effectively removed both TCE and MTBE from aqueous solution than hydrophilic adsorbents, a result that was explained by enhanced water adsorption on hydrophilic surfaces. To assure sufficient adsorbent hydrophobicity, the oxygen and nitrogen contents of an activated carbon should therefore sum to no more than about 2 to 3 mmol/g.

Membrane insertion: The strategies of toxins (review).

Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells. They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm. As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane. Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis. In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, i.e. it contains hydrophobic and amphipathic alpha-helices long enough to span a membrane. To render this 'membrane protein' water-soluble during the initial part of its life the hydrophobic helices are sheltered from the solvent by a barrel of amphipathic helices. In the second group of toxins the opposite strategy is adopted. The toxin is an intrinsically soluble protein and is composed mainly of beta-structure. These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic beta-sheet to generate sufficient hydrophobicity for membrane insertion to occur. Toxins from this latter group are thought to perforate the lipid bilayer as a beta-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal alpha-toxin. The two groups of toxins will be described in detail through the presentation of examples. Particular attention will be given to the beta-structure toxins, since four new structures have been solved over the past year: the staphyloccocal alpha-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin. Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed. Peptide toxins will not be covered in this review.

Reducing-End Modification of N-Linked Oligosaccharides with Tyrosine

The N-linked oligosaccharides from bovine fetuin were purified using newly developed preparative purification methodology. N-linked oligosaccharides were released from tryptic glycopeptides utilizing N-glycosidase F on the 5-g scale. Selective desialylation with neuraminidase from Clostridium perfringens resulted in the formation of a mono-sialyl-oligosaccharide and asialo-oligosaccharides. The reducing ends of the oligosaccharides were converted to the glycosylamine and reacted with the N-hydroxysuccinimide ester of Boctyrosine. The tyrosinated oligosaccharides were resolved into individual peaks on RP-HPLC and then characterized by proton NMR and FAB-MS. A single asialo-triantennary, an asialo-biantennary, and a mono-sialyl-triantennary oligosaccharide were recovered in good yield. Each product contained a single BocTyr residue attached to the reducing-end GlcNAc residue through a β-glycosylamide linkage. The procedure was utilized to isolate multi-micromole quantities of oligosaccharides from gram quantities of glycoprotein, thus providing a new route to purify large quantities of N-linked oligosaccharides which contain a terminal tyrosine residue. The tyrosinated oligosaccharides are valuable glycoconjugate ligands which contain a chromophore that absorbs at 280 nm and has sufficient hydrophobicity to facilitate RP-HPLC separations. Furthermore, this group can be deprotected by removal of Hoc to reveal a primary amine suitable for further derivatization and can also be radioiodinated for tracking during biological experiments.

Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.