The components of biological membranes are asymmetrically distributed between the membrane surfaces. Proteins are absolutely asymmetrical in that every copy of a polypeptide chain has the same orientation in the membrane, and lipids are nonabsolutely asymmetrical in that almost every type of lipid is present on both sides of the bilayer, but in different and highly variable amounts. Asymmetry is maintained by lack of transmembrane diffusion. Two types of membrane proteins, called ectoproteins and endoproteins, are distinguished. Biosynthetic pathways for both types of proteins and for membrane lipids are inferred from their topography and distribution in the formed cells. Note added in proof. A cell-free system has now been developed which permits the mechanisms of membrane protein assembly to be studied (108). The membrane glycoprotein of vesicular stomatitis virus has been synthesized by wheat germ ribosomes in the presence of rough endoplasmic reticulum from pancreas. The resulting polypeptide is incorporated into the membrane, spans the lipid bilayer asymmetrically, and is glycosylated (108). The amino terminal portion of this transmembrane protein is found inside the endoplasmic reticulum vesicle, while the carboxyl terminal portion is exposed on the outer surface of the vesicle. Furthermore, addition of the glycoprotein to membranes after protein synthesis does not result in incorporation of the protein into the membrane in the manner described above (108). Consequently, protein synthesis and incorporation into the membrane must be closely coupled. Indeed, using techniques to synchronize the growth of nascent polypeptides, it has been shown (109) that no more than one-fourth of the glycoprotein chain can be made in the absence of membranes and still cross the lipid bilayer when chains are subsequently completed in the presence of membranes. These findings demonstrate directly that the extracytoplasmic portion of an ectoprotein can cross the membrane only during biosynthesis, and not after.
The transmembrane (TM) domains of viral fusion proteins are required for fusion, but their precise role is unknown. G protein, the fusion protein of vesicular stomatitis virus, was previously shown to lose syncytia-forming ability if six residues (GLIIGL) were deleted from its TM domain. The 20-residue TM domain of wild-type (TM20) G protein was thus changed into a TM domain of 14 residues (TM14). To assess possible sequence specificity for this loss of function, the two Gly residues in TM20 were replaced with either Ala or Leu. Both mutations resulted in complete loss of fusion activity, as measured by fusion-dependent reporter gene transfer. Single substitutions decreased activity by about half. TM14 was weakly active (15%) but reintroduction of a Gly residue into TM14 by a single Ile 3 Gly substitution increased activity to 80%. All mutants retained normal hemifusion activity, i.e., lipid mixing between the outer leaflets of the reacting membranes. Thus, at least one TM Gly residue is required for a late step in fusion mediated by G protein. Gly residues were significantly (2.6-fold; P ؍ 0.004) more abundant in the TM domains of viral fusion proteins than in those of nonfusion proteins and were distributed differently within the TM domain. Thus, Gly residues in the TM domain of other viral fusion proteins may also prove to be important for fusion activity.
The mechanism of the antiviral activity of hypericin was characterized and compared with that of rose bengal. Both compounds inactivate enveloped (but not unenveloped) viruses upon illumination by visible light. Human immunodeficiency and vesicular stomatitis viruses were photodynamically inactivated by both dyes at nanomolar concentrations. Photodynamic inactivation of fusion (hemolysis) by vesicular stomatitis, influenza, and Sendai viruses was induced by both dyes under similar conditions (e.g., I50 = 20-50 nM for vesicular stomatitis virus), suggesting that loss of infectivity resulted from inactivation of fusion. Syncytium formation, between cells activated to express human immunodeficiency virus gpl20 on their surfaces and CD4+ cells, was inhibited by illumination in the presence of 1 ,uM hypericin. Hypericin and rose bengal thus exert similar virucidal effects. Both presumably act by the same mechanism-namely, the inactivation of the viral fusion function by singlet oxygen produced upon illumination. The implications of this photodynamic antiviral action for the potential therapeutic usefulness of both hypericin and rose bengal are discussed.
Casein kinase‐II (CK‐II) is a widely distributed protein kinase, which plays numerous roles in the regulation of transcription through modification of transacting transcription factors. Phosphorylation of vesicular stomatitis virus (VSV) P protein by CK‐II was found to be both necessary and sufficient for transcriptional activation. Upon treatment of P by CK‐II, activity was acquired faster (t1/2 = 3.7 min) than were total phosphates (t1/2 = 7.4 min). Stoichiometry was 2 mol phosphate/mol P, indicating activation by phosphorylation at either one or both of two independent sites. The sites were identified by substituting aspartate (D) residues at either S60 or T62, producing proteins that were partly active without phosphorylation, but were fully active at higher concentrations; CK‐II added only a single phosphate group to each of these, and conferred full activity. P protein doubly substituted with D at S60 and T62 was fully active without phosphorylation, and was not a substrate for CK‐II. Active P protein, whether CK‐II treated or doubly substituted, was shown by gel filtration and crosslinking to exist as a discretely multimeric, probably tetrameric, structure. The singly substituted mutants were partly multimeric, becoming fully so after CK‐II treatment. Phosphorylation by CK‐II thus mediates the self‐association of P into the multimeric, transcriptionally active form.
Caenorhabditis elegans requires sterol, usually supplied as cholesterol, but this is enzymatically modified, and different sterols can substitute. Sterol deprivation decreased brood size and adult growth in the first generation, and completely, reversibly, arrested growth as larvae in the second. After one generation of sterol deprivation, 10 ng/ml cholesterol allowed delayed laying of a few eggs, but full growth required 300 ng/ml. C. elegans synthesizes two unusual 4 ␣ -methyl sterols (4MSs), but each 4MS supported only limited growth as the sole sterol. However, addition of only 10 ng of cholesterol to 1,000 ng of 4MS restored full growth and egg-laying, suggesting that both a 4MS and an unmethylated sterol are required for development. Filipin stained sterols in only a few specific cells: the excretory gland cell, two amphid socket cells, two phasmid socket cells and, in males, spicule socket cells. Sterols were also present in the pharynx and in the intestine of feeding animals in a proximal-to-distal gradient.This non-random sterol distribution, the low concentration requirements, and the effects of 4MSs argues that sterols are unlikely to be used for bulk structural modification of cell membranes, but may be required as hormone precursors and/or developmental effectors. Dietary sterol is required by Caenorhabditis elegans (1, 2) because, like insects, C. elegans is incapable of synthesizing the four-ring sterol nucleus, but its functions remain largely unknown. The existence of sterol-based hormones in C. elegans has recently been suggested, but no hormone has yet been identified (3, 4). Cholesterol is known to be extensively metabolized by C. elegans to form several other sterols, including two unusual 4 ␣ -methyl sterols (4MSs), which are present in substantial amounts (5-8, 9). These sterols might thus be functional, instead of or in addition to cholesterol itself.Insects resemble these nematodes in requiring sterols but being unable to synthesize them. Two functions for cholesterol are known in insects: as the metabolic precursor of the molting hormone ecdysone (10), and as the moiety required for activation by covalent attachment to the morphogen protein hedgehog (11). Insect cells, unlike vertebrate cells, grow normally under sterol-free conditions, and thus do not need cholesterol in their plasma membranes (12)(13)(14).We have now characterized the sterol requirements of C. elegans in some detail. Conditions for stringent sterol deprivation were developed, and the consequences are described. The use of these conditions allowed us to investigate minimum cholesterol requirements, and the ability of other sterols to substitute. Partial and synergistic effects were found, suggesting that different sterols have diverse effects mediated by several pathways. The accumulation of sterol in the intestinal tract and in a few specific cells in C. elegans was also demonstrated by filipin staining, which stains all 3  -hydroxy sterols.These observations provide a basis for a comprehensive study of ster...
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