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Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

Gadelha C, Zhang W, Chamberlain JW, Chait BT, Wickstead B, Field MC - Mol. Cell Proteomics (2015)

Bottom Line: This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival.Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket.This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis.

View Article: PubMed Central - PubMed

Affiliation: From the ‡School of Life Sciences, University of Nottingham, Nottingham, UK, NG2 7UH; §Department of Pathology, University of Cambridge, Cambridge, UK, CB2 1QP; catarina.gadelha@nottingham.ac.uk.

No MeSH data available.


Related in: MedlinePlus

Most ESAGs encode surface-associated proteins. 9 ESAGs were localized by tagging the respective gene at the active ES (except for ESAG5, 10 and 11, for which a surface proteome GRESAG was used) with an ORF encoding superfolder GFP and imaged using native fluorescence microscopy. Signal from superfolder-GFP is shown in yellow. Cells have been counterstained with concanavalin A (ConA, blue) and DAPI (magenta). The FP is indicated by yellow arrowhead.
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Figure 7: Most ESAGs encode surface-associated proteins. 9 ESAGs were localized by tagging the respective gene at the active ES (except for ESAG5, 10 and 11, for which a surface proteome GRESAG was used) with an ORF encoding superfolder GFP and imaged using native fluorescence microscopy. Signal from superfolder-GFP is shown in yellow. Cells have been counterstained with concanavalin A (ConA, blue) and DAPI (magenta). The FP is indicated by yellow arrowhead.

Mentions: Chimeric proteins were created by integration of tagging constructs at endogenous gene loci. Tagging cell surface proteins is potentially complicated by requirements for signaling sequences at both amino and carboxyl termini, and issues with folding of fluorescent proteins targeted through the ER. To overcome these problems, we created two new series of vectors specifically designed for the endogenous-locus tagging of genes encoding GPI-anchored and non-GPI-anchored sequences containing N-terminal signal sequences. These vectors are called the pSiG and pSiS series, respectively (supplemental Fig. S2) and include the incorporation of a “superfolder” GFP (or derivatives) with improved folding dynamics and greater resistance to the reducing environments encountered in the ER lumen or extracellular space compared with conventional GFP variants (34), plus an epitope tag (HA). The pSiG/pSiS series also include processing signals (trypanosome signal peptide or GPI-anchor addition sequences), providing a means to rapidly and accurately tag surface proteins at either N- or C terminus (supplemental Fig. S2). These vectors provide the correct FP localization of previously analyzed proteins, for example, either GPI-anchored or non-anchored subunits of the transferrin receptor (see Fig. 7). Moreover, the toolkit does not force a non-TbBSP protein (ESAG1) onto the cell surface (supplemental Fig. S2).


Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

Gadelha C, Zhang W, Chamberlain JW, Chait BT, Wickstead B, Field MC - Mol. Cell Proteomics (2015)

Most ESAGs encode surface-associated proteins. 9 ESAGs were localized by tagging the respective gene at the active ES (except for ESAG5, 10 and 11, for which a surface proteome GRESAG was used) with an ORF encoding superfolder GFP and imaged using native fluorescence microscopy. Signal from superfolder-GFP is shown in yellow. Cells have been counterstained with concanavalin A (ConA, blue) and DAPI (magenta). The FP is indicated by yellow arrowhead.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587319&req=5

Figure 7: Most ESAGs encode surface-associated proteins. 9 ESAGs were localized by tagging the respective gene at the active ES (except for ESAG5, 10 and 11, for which a surface proteome GRESAG was used) with an ORF encoding superfolder GFP and imaged using native fluorescence microscopy. Signal from superfolder-GFP is shown in yellow. Cells have been counterstained with concanavalin A (ConA, blue) and DAPI (magenta). The FP is indicated by yellow arrowhead.
Mentions: Chimeric proteins were created by integration of tagging constructs at endogenous gene loci. Tagging cell surface proteins is potentially complicated by requirements for signaling sequences at both amino and carboxyl termini, and issues with folding of fluorescent proteins targeted through the ER. To overcome these problems, we created two new series of vectors specifically designed for the endogenous-locus tagging of genes encoding GPI-anchored and non-GPI-anchored sequences containing N-terminal signal sequences. These vectors are called the pSiG and pSiS series, respectively (supplemental Fig. S2) and include the incorporation of a “superfolder” GFP (or derivatives) with improved folding dynamics and greater resistance to the reducing environments encountered in the ER lumen or extracellular space compared with conventional GFP variants (34), plus an epitope tag (HA). The pSiG/pSiS series also include processing signals (trypanosome signal peptide or GPI-anchor addition sequences), providing a means to rapidly and accurately tag surface proteins at either N- or C terminus (supplemental Fig. S2). These vectors provide the correct FP localization of previously analyzed proteins, for example, either GPI-anchored or non-anchored subunits of the transferrin receptor (see Fig. 7). Moreover, the toolkit does not force a non-TbBSP protein (ESAG1) onto the cell surface (supplemental Fig. S2).

Bottom Line: This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival.Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket.This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis.

View Article: PubMed Central - PubMed

Affiliation: From the ‡School of Life Sciences, University of Nottingham, Nottingham, UK, NG2 7UH; §Department of Pathology, University of Cambridge, Cambridge, UK, CB2 1QP; catarina.gadelha@nottingham.ac.uk.

No MeSH data available.


Related in: MedlinePlus