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PMID: 9736719 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 95 ·No. 19 ·1998-09-15 ·Pages 11235-40

Eskandari S, Wright EM, Kreman M, Starace DM, Zampighi GA

Abstract

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with alpha-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 +/- 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 +/- 2 transmembrane helices, connexin 50 is a hexamer containing 24 +/- 3 helices, the major intrinsic protein is a tetramer containing 24 +/- 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 +/- 2 helices.

MeSH Terms
Animals Aquaporin 1 Aquaporins/ultrastructure Cell Membrane/chemistry Connexins/ultrastructure Cystic Fibrosis Transmembrane Conductance Regulator/ultrastructure Eye Proteins/ultrastructure Freezing Ion Channels/ultrastructure Membrane Glycoproteins/ultrastructure Membrane Proteins/ultrastructure Microscopy, Electron Monosaccharide Transport Proteins/ultrastructure Oocytes/chemistry Particle Size Protein Conformation Protein Structure, Secondary Recombinant Proteins/ultrastructure Sodium-Glucose Transporter 1 Xenopus laevis
Chemicals
Aquaporins Connexins Eye Proteins Ion Channels Membrane Glycoproteins Membrane Proteins Monosaccharide Transport Proteins Recombinant Proteins Sodium-Glucose Transporter 1 aquaporin 0 Cystic Fibrosis Transmembrane Conductance Regulator Aquaporin 1
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Eskandari S
Department of Physiology, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095-1751, USA. [email protected]
Wright E M
Kreman M
Starace D M
Zampighi G A
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.95.19.11235
Email
[email protected]
Published
1998-09-15
Pages
11235-40
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC21625
Subset
IM
Grants
NIDDK NIH HHS · R01 DK019567 · United States
NEI NIH HHS · EY-04110 · United States
NINDS NIH HHS · NS-25554 · United States
NIDDK NIH HHS · DK-19567 · United States
NEI NIH HHS · R01 EY004110 · United States
External Links
PubMed source DOI: 10.1073/pnas.95.19.11235 PMC Full Text View journal details
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