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Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes.

Pieper R, Huang ST, Clark DJ, Robinson JM, Alami H, Parmar PP, Suh MJ, Kuntumalla S, Bunai CL, Perry RD, Fleischmann RD, Peterson SN - Proteome Sci (2009)

Bottom Line: Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries).Thirty-one proteins were experimentally associated with the outer membrane (OM).The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six beta-barrel OM protein families and 25 distinct IM transporter families.

View Article: PubMed Central - HTML - PubMed

Affiliation: J. Craig Venter Institute, Rockville, Maryland, USA. rpieper@jcvi.org

ABSTRACT
Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six beta-barrel OM protein families and 25 distinct IM transporter families.

No MeSH data available.


Related in: MedlinePlus

A comparison of spot profiles in 2D gels derived from a Y. pestis KIM6+ whole cell lysate and five subcellular fractions. Acronyms are described in the flowchart of Figure 1. Cells were grown to stationary phase at 26°C. First dimension IEF separations were performed in the pH range from 4 to 7. The Mr range of second dimension SDS-PAGE separations was 10–200 kDa. Gel image analysis details are provided in the text. Spot identifications by MS confirmed appropriate spot matching. Spot numbers are equivalent to those denoted in Table 1; Additional File 2.
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Figure 3: A comparison of spot profiles in 2D gels derived from a Y. pestis KIM6+ whole cell lysate and five subcellular fractions. Acronyms are described in the flowchart of Figure 1. Cells were grown to stationary phase at 26°C. First dimension IEF separations were performed in the pH range from 4 to 7. The Mr range of second dimension SDS-PAGE separations was 10–200 kDa. Gel image analysis details are provided in the text. Spot identifications by MS confirmed appropriate spot matching. Spot numbers are equivalent to those denoted in Table 1; Additional File 2.

Mentions: Proteins enriched in hs-MBR and hpH-MBR fractions, but also detected in soluble CYP or PPS fractions, comprised 99 entries in Additional File 2 (p-M category). In quantitative terms, EM and EIM values fell into the ranges of 0.05 to 1.2 and 0.3 to 4, respectively. Their quantitative distribution among membrane fractions is compatible with the general definition of peripheral membrane proteins. These proteins appeared to associate with membranes temporarily, did not require detergents for solubilization and were also present in cytoplasmic and/or periplasmic fractions. High salt and high pH membrane extraction conditions favor the solubilization of proteins bound to membranes electrostatically. Judged from their abundance in CYP vs. PPS fractions and the presence of export signal sequences, most of these proteins should localize at the cytoplasmic surface of the IM. Many of these proteins are also predicted to be peripheral membrane components of multi-subunit complexes and associate with other integral membrane proteins. Examples are NuoC (#71), LpdA (#92), ManX (#101), AdhE (#99), AtpD (#110), AtpA (#127) and GuaB (#164). All protein numbers referenced here and in the following two sections pertain to Figure 3, unless otherwise stated. Such protein complexes, often characterized in E. coli, include ATP synthase (AtpA and AtpD), NADH dehydrogenase (NuoC), pyruvate dehydrogenase (LpdA) and a mannose-specific phosphotransferase system (ManX).


Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes.

Pieper R, Huang ST, Clark DJ, Robinson JM, Alami H, Parmar PP, Suh MJ, Kuntumalla S, Bunai CL, Perry RD, Fleischmann RD, Peterson SN - Proteome Sci (2009)

A comparison of spot profiles in 2D gels derived from a Y. pestis KIM6+ whole cell lysate and five subcellular fractions. Acronyms are described in the flowchart of Figure 1. Cells were grown to stationary phase at 26°C. First dimension IEF separations were performed in the pH range from 4 to 7. The Mr range of second dimension SDS-PAGE separations was 10–200 kDa. Gel image analysis details are provided in the text. Spot identifications by MS confirmed appropriate spot matching. Spot numbers are equivalent to those denoted in Table 1; Additional File 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2663777&req=5

Figure 3: A comparison of spot profiles in 2D gels derived from a Y. pestis KIM6+ whole cell lysate and five subcellular fractions. Acronyms are described in the flowchart of Figure 1. Cells were grown to stationary phase at 26°C. First dimension IEF separations were performed in the pH range from 4 to 7. The Mr range of second dimension SDS-PAGE separations was 10–200 kDa. Gel image analysis details are provided in the text. Spot identifications by MS confirmed appropriate spot matching. Spot numbers are equivalent to those denoted in Table 1; Additional File 2.
Mentions: Proteins enriched in hs-MBR and hpH-MBR fractions, but also detected in soluble CYP or PPS fractions, comprised 99 entries in Additional File 2 (p-M category). In quantitative terms, EM and EIM values fell into the ranges of 0.05 to 1.2 and 0.3 to 4, respectively. Their quantitative distribution among membrane fractions is compatible with the general definition of peripheral membrane proteins. These proteins appeared to associate with membranes temporarily, did not require detergents for solubilization and were also present in cytoplasmic and/or periplasmic fractions. High salt and high pH membrane extraction conditions favor the solubilization of proteins bound to membranes electrostatically. Judged from their abundance in CYP vs. PPS fractions and the presence of export signal sequences, most of these proteins should localize at the cytoplasmic surface of the IM. Many of these proteins are also predicted to be peripheral membrane components of multi-subunit complexes and associate with other integral membrane proteins. Examples are NuoC (#71), LpdA (#92), ManX (#101), AdhE (#99), AtpD (#110), AtpA (#127) and GuaB (#164). All protein numbers referenced here and in the following two sections pertain to Figure 3, unless otherwise stated. Such protein complexes, often characterized in E. coli, include ATP synthase (AtpA and AtpD), NADH dehydrogenase (NuoC), pyruvate dehydrogenase (LpdA) and a mannose-specific phosphotransferase system (ManX).

Bottom Line: Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries).Thirty-one proteins were experimentally associated with the outer membrane (OM).The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six beta-barrel OM protein families and 25 distinct IM transporter families.

View Article: PubMed Central - HTML - PubMed

Affiliation: J. Craig Venter Institute, Rockville, Maryland, USA. rpieper@jcvi.org

ABSTRACT
Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six beta-barrel OM protein families and 25 distinct IM transporter families.

No MeSH data available.


Related in: MedlinePlus