Limits...
Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei.

Colasante C, Voncken F, Manful T, Ruppert T, Tielens AG, van Hellemond JJ, Clayton C - F1000Res (2013)

Bottom Line: The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3.However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found.We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut für Anatomie und Zellbiologie, Giessen, 35392, Germany.

ABSTRACT
In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

No MeSH data available.


Immunofluorescence analysis of the putative glycosomal membrane proteins in procyclicT. brucei.N-terminally or C-terminally myc-tagged versions of the proteins were detected (green) by using a commercial Myc antibody (Sigma-Aldrich). Mitochondria were visualized (red) using mitotracker. The endoplasmatic reticulum (ER) was detected using an antibody directed against the ER lumen protein BiP (red). Overlays (Merge) of the green staining and the red staining are shown to visualize the common compartmentalization of the proteins. On the right side, western blots are shown to illustrate the expression of the myc-tagged membrane proteins. (+) and (-) indicate tetracycline-induced and -uninduced cells respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3814921&req=5

SF1: Immunofluorescence analysis of the putative glycosomal membrane proteins in procyclicT. brucei.N-terminally or C-terminally myc-tagged versions of the proteins were detected (green) by using a commercial Myc antibody (Sigma-Aldrich). Mitochondria were visualized (red) using mitotracker. The endoplasmatic reticulum (ER) was detected using an antibody directed against the ER lumen protein BiP (red). Overlays (Merge) of the green staining and the red staining are shown to visualize the common compartmentalization of the proteins. On the right side, western blots are shown to illustrate the expression of the myc-tagged membrane proteins. (+) and (-) indicate tetracycline-induced and -uninduced cells respectively.

Mentions: By comparison with the predicted proteome ofL. tarentolae24, 464 polypeptides were identified (Supplementary Table S1). The first step that we undertook was to identify homologues of all identified proteins from theT. brucei genome (http://tritrypdb.org/tritrypdb/). This was done to facilitate the retrieval of information because most experimental data is available exclusively forT. brucei. All identified proteins were screened for database annotation, including user comments, and in some cases we also updated annotations from publications. We further screened all proteins for their presence in previously published glycosomal1,10 and mitochondrial34 proteomes. The results are summarised inSupplementary Table S1, Sheet 1. Proteins that were clearly located in compartments other than the glycosome were then excluded, resulting inSupplementary Table S1, Sheet 2. Some candidates predicted to contain at least one trans-membrane domain were tested for their locations, by expression of N-terminally and/or C-terminally tagged versions (none has a PTS1 signal). The proteins encoded by Tb927.3.1840 (putative 3-oxo-5-alpha-steroid 4-dehydrogenase), Tb927.5.1210 (putative short-chain dehydrogenase) and Tb927.10.14020 (unknown function) were all targeted to mitochondria, while Tb927.7.3900 (annotated as a vacuolar transporter chaperone) was in the ER(Supplementary Figure S1). All identified proteins were further searched for the presence of known peroxisomal targeting signals in theL. major orT. brucei homologues35; in addition, theL. tarentolae protein sequences inSupplementary Table S1, Sheet 2 were manually scanned for PTS1 signals.


Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei.

Colasante C, Voncken F, Manful T, Ruppert T, Tielens AG, van Hellemond JJ, Clayton C - F1000Res (2013)

Immunofluorescence analysis of the putative glycosomal membrane proteins in procyclicT. brucei.N-terminally or C-terminally myc-tagged versions of the proteins were detected (green) by using a commercial Myc antibody (Sigma-Aldrich). Mitochondria were visualized (red) using mitotracker. The endoplasmatic reticulum (ER) was detected using an antibody directed against the ER lumen protein BiP (red). Overlays (Merge) of the green staining and the red staining are shown to visualize the common compartmentalization of the proteins. On the right side, western blots are shown to illustrate the expression of the myc-tagged membrane proteins. (+) and (-) indicate tetracycline-induced and -uninduced cells respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3814921&req=5

SF1: Immunofluorescence analysis of the putative glycosomal membrane proteins in procyclicT. brucei.N-terminally or C-terminally myc-tagged versions of the proteins were detected (green) by using a commercial Myc antibody (Sigma-Aldrich). Mitochondria were visualized (red) using mitotracker. The endoplasmatic reticulum (ER) was detected using an antibody directed against the ER lumen protein BiP (red). Overlays (Merge) of the green staining and the red staining are shown to visualize the common compartmentalization of the proteins. On the right side, western blots are shown to illustrate the expression of the myc-tagged membrane proteins. (+) and (-) indicate tetracycline-induced and -uninduced cells respectively.
Mentions: By comparison with the predicted proteome ofL. tarentolae24, 464 polypeptides were identified (Supplementary Table S1). The first step that we undertook was to identify homologues of all identified proteins from theT. brucei genome (http://tritrypdb.org/tritrypdb/). This was done to facilitate the retrieval of information because most experimental data is available exclusively forT. brucei. All identified proteins were screened for database annotation, including user comments, and in some cases we also updated annotations from publications. We further screened all proteins for their presence in previously published glycosomal1,10 and mitochondrial34 proteomes. The results are summarised inSupplementary Table S1, Sheet 1. Proteins that were clearly located in compartments other than the glycosome were then excluded, resulting inSupplementary Table S1, Sheet 2. Some candidates predicted to contain at least one trans-membrane domain were tested for their locations, by expression of N-terminally and/or C-terminally tagged versions (none has a PTS1 signal). The proteins encoded by Tb927.3.1840 (putative 3-oxo-5-alpha-steroid 4-dehydrogenase), Tb927.5.1210 (putative short-chain dehydrogenase) and Tb927.10.14020 (unknown function) were all targeted to mitochondria, while Tb927.7.3900 (annotated as a vacuolar transporter chaperone) was in the ER(Supplementary Figure S1). All identified proteins were further searched for the presence of known peroxisomal targeting signals in theL. major orT. brucei homologues35; in addition, theL. tarentolae protein sequences inSupplementary Table S1, Sheet 2 were manually scanned for PTS1 signals.

Bottom Line: The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3.However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found.We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut für Anatomie und Zellbiologie, Giessen, 35392, Germany.

ABSTRACT
In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

No MeSH data available.