Assembly of Multisubunit Membrane Proteins

Carlin, B E; Merlie, John P.

doi:10.1007/978-1-4612-4930-6_5

Cited by 23 publications

(30 citation statements)

References 96 publications

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“…Oligomerization of membrane proteins plays a role in their transport through both the endocytic and biosynthetic pathways (Carlin and Merlie, 1986;Mellman, 1987). The IgG Fc receptor, for example, is rapidly internalized and delivered to lysosomes only when cross-linked into a higher-order structure (Ukkonen et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

“…The average amount of proteins pelleting to the bottom of the tube is shown (T), and was determined as described in Fig. 2. have been identified, which apparently acquire their final quaternary structure shortly after biosynthesis (Bole et al, 1986;Carlin and Merlie, 1986;Kreis and Lodish, 1986). We have found that two well-characterized viral spike glycoproteins, the influenza HA and the VSV G protein, form trimers in the ER (Doms et al, 1987;Copeland et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

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Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein.

Doms

Ruusala

Machamer

et al. 1988

The Journal of Cell Biology

193

148

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Abstract. The vesicular stomatitis virus glycoprotein (G protein) is an integral membrane protein which assembles into noncovalently associated trimers before transport from the endoplasmic reticulum. In this study we have examined the folding and oligomeric assembly of twelve mutant G proteins with alterations in the cytoplasmic, transmembrane, or ectodomains. Through the use of conformation-specific antibodies, we found that newly synthesized G protein folded into a conformation similar to the mature form within 1-3 min of synthesis and before trimer formation. Mutant proteins not capable of undergoing correct initial folding did not trimerize, were not transported, and were found in large aggregates. They had, as a rule, mutations in the ectodomain, including several with altered glycosylation patterns. In contrast, mutations in the cytoplasmic domain generally had little effect on folding and trimerization. These mutant proteins, whose ectodomains were identical to the wild-type by several assays, were either transported to the cell surface slowly or not at all. We concluded that while correct ectodomain folding and trimer formation are prerequisites for transport, they alone are not sufficient. The results suggest that the cytoplasmic domain of the wild-type protein may facilitate rapid, efficient transport from the ER, which can be easily affected or eliminated by tail mutations that do not detectably affect the ectodomain.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein.

Doms

Ruusala

Machamer

et al. 1988

The Journal of Cell Biology

193

148

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“…Protein oligomerization adds further complexity to protein folding and secretion and was one basis for believing that eukaryotic expression might be advantageous over bacterial expression [see review by ]. Oligomeric proteins typically assemble in the ER , and proper assembly can take between 5 min to 2 h (Carlin and Merlie, 1986;Olson and Lane, 1987;Doms et al, 1988). At this point, we have not ruled out the possibility that tetrameric misassociation may be involved in ER retention.…”

Section: Discussionmentioning

confidence: 95%

Overexpression of an archaeal protein in yeast: Secretion bottleneck at the ER

Smith¹,

Robinson²

2002

Biotech & Bioengineering

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Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity. We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture. All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields.

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“…Fitting the data to a Bleany-Bowers equation [38], gives a separation of +10 K (7 cm-I ) between the magnetic ground and excited states. Spectroelectrochemical studies evidenced the lack of any intervalence band transition originated by the presence of an lET phenomenon.…”

Section: Synthesis Of a Metallocene Snbstituted Diradicalmentioning

confidence: 99%