Glucose Transporter Oligomeric Structure Determines the Mechanism of Glucose Transport: A Dissertation

The relationship between human erythrocyte glucose transporter (GLUT1) oligomeric structure and function was studied. GLUT1 was purified from human erythrocytes in the absence (GLUT1-DTT) or the presence (GLUT1+DTT) of dithiothreitol. Chemical cross-linking studies of lipid bilayer-resident purified GLUT1 and hydrodynamic studies of cholate-solubilized GLUT1 support the view that GLUT1-DTT is a homotetramer and GLUT1+DTT is a homodimer. Parallel studies on human erythrocyte, and studies employing conformation-specific antibodies (anti-GLUT1-DTT antibodies, ∂-IgGs), indicate that erythrocyte-resident GLUT1 resembles GLUT1-DTT (a homotetramer). While the D-glucose binding capacities of GLUT1-DTT and GLUT1+DTT are indistinguishable, GLUT1-DTT presents at least two population of binding sites to D-glucose whereas GLUT1+DTT presents only one population of sugar binding sites. The cytochalasin B (CCB) binding capacity of GLUT1-DTT (0.4 sites/monomer) is one half of that of GLUT1+DTT. GLUT1-DTT and GLUT1+DTT contain 2 and 6 free sulfhydryls per monomer respectively. The subunits (monomers) of tetrameric and dimeric GLUT1 are not linked by disulfide bridges. Erythrocyte resident GLUT1 presents at least two binding sites to D-glucose and binds CCB with a molar stoichiometry of 0.55 sites per GLUT1 monomer. Following treatment with high pH plus dithiothreitol, the sugar binding capacity of erythrocyte membrane resident transporter is unaltered but the transporter now presents only one population of binding sites to D-glucose, binds CCB with molar stoichiometry of 1.3 sites per GLUT1 monomer and displays significantly reduced affinity for ∂-IgGs. These findings demonstrate that erythrocyte resident glucose transporter is GLUT1-DTT (a GLUT1 tetramer) and that GLUT1 oligomeric structure determines GLUT1 functional properties. A model which rationalizes these findings is proposed

Protein Translocons Multifunctional Mediators of Protein Translocation across Membranes

AbstractProtein translocation systems consist of complex molecular machines whose activities are not limited to unidirectional protein targeting. Protein translocons and their associated receptor systems can be viewed as dynamic modular units whose interactions, and therefore functions, are regulated in response to specific signals. This flexibility allows translocons to interact with multiple signal receptor systems to manage the targeting of topologically distinct classes of proteins, to mediate targeting to different suborganellar compartments, and to respond to stress and developmental cues. Furthermore, the activities of translocons are tightly coordinated with downstream events, thereby providing a direct link between targeting and protein maturation

A Very Late Viral Protein Triggers the Lytic Release of SV40

How nonenveloped viruses such as simian virus 40 (SV40) trigger the lytic release of their progeny is poorly understood. Here, we demonstrate that SV40 expresses a novel later protein termed VP4 that triggers the timely lytic release of its progeny. Like VP3, VP4 synthesis initiates from a downstream AUG start codon within the VP2 transcript and localizes to the nucleus. However, VP4 expression occurs ∼24 h later at a time that coincides with cell lysis, and it is not incorporated into mature virions. Mutation of the VP4 initiation codon from the SV40 genome delayed lysis by 2 d and reduced infectious particle release. Furthermore, the co-expression of VP4 and VP3, but not their individual expression, recapitulated cell lysis in bacteria. Thus, SV40 regulates its life cycle by the later temporal expression of VP4, which results in cell lysis and enables the 50-nm virus to exit the cell. This study also demonstrates how viruses can generate multiple proteins with diverse functions and localizations from a single reading frame

A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions

Evaluation of Adrenal Suppression of a Lipid Enhanced, Topical Emollient Cream Formulation of Hydrocortisone Butyrate 0.1% in Treating Children with Atopic Dermatitis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75102/1/j.1525-1470.2007.00342.x.pd

EDEM1\u27s mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase–like 1 protein (EDEM1) is a protein quality control factor that was initially proposed to recognize N-linked glycans on misfolded proteins through its mannosidase-like domain (MLD). However, recent studies have demonstrated that EDEM1 binds to some misfolded proteins in a glycan-independent manner, suggesting a more complex binding landscape for EDEM1. In this study, we have identified a thiol-dependent substrate interaction between EDEM1 and the α1-antitrypsin ER-associated protein degradation (ERAD) clients Z and NHK, specifically through the single Cys residue on Z/NHK (Cys256), required for binding under stringent detergent conditions. In addition to the thiol-dependent interaction, the presence of weaker protein-protein interactions was confirmed, suggestive of bipartite client-binding properties. About four reactive thiols on EDEM1 were identified and were not directly responsible for the observed redox-sensitive binding by EDEM1. Moreover, a protein construct comprising the EDEM1 MLD had thiol-dependent binding properties along with its active glycan-trimming activities. Lastly, we identified an additional intrinsically disordered region (IDR) located at the C terminus of EDEM1 in addition to its previously identified N-terminal IDR. We also determined that both IDRs are required for binding to the ERAD component ERdj5 as an interaction with ERdj5 was not observed with the MLD alone. Together, our findings indicate that EDEM1 employs different binding modalities to interact with ERAD clients and ER quality control (ERQC) machinery partners and that some of these properties are shared with its homologues EDEM2 and EDEM3

The role of UDP-Glc:glycoprotein glucosyltransferase 1 in the maturation of an obligate substrate prosaposin

A natural substrate for UGT1 is confirmed, revealing how the enzyme functions in the calnexin chaperone system as a quality control step in protein folding

Reading the Complex Skipper Butterfly Fauna of One Tropical Place

BACKGROUND: An intense, 30-year, ongoing biodiversity inventory of Lepidoptera, together with their food plants and parasitoids, is centered on the rearing of wild-caught caterpillars in the 120,000 terrestrial hectares of dry, rain, and cloud forest of Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. Since 2003, DNA barcoding of all species has aided their identification and discovery. We summarize the process and results for a large set of the species of two speciose subfamilies of ACG skipper butterflies (Hesperiidae) and emphasize the effectiveness of barcoding these species (which are often difficult and time-consuming to identify). METHODOLOGY/PRINCIPAL FINDINGS: Adults are DNA barcoded by the Biodiversity Institute of Ontario, Guelph, Canada; and they are identified by correlating the resulting COI barcode information with more traditional information such as food plant, facies, genitalia, microlocation within ACG, caterpillar traits, etc. This process has found about 303 morphologically defined species of eudamine and pyrgine Hesperiidae breeding in ACG (about 25% of the ACG butterfly fauna) and another 44 units indicated by distinct barcodes (n = 9,094), which may be additional species and therefore may represent as much as a 13% increase. All but the members of one complex can be identified by their DNA barcodes. CONCLUSIONS/SIGNIFICANCE: Addition of DNA barcoding to the methodology greatly improved the inventory, both through faster (hence cheaper) accurate identification of the species that are distinguishable without barcoding, as well as those that require it, and through the revelation of species "hidden" within what have long been viewed as single species. Barcoding increased the recognition of species-level specialization. It would be no more appropriate to ignore barcode data in a species inventory than it would be to ignore adult genitalia variation or caterpillar ecology