Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.
Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala(140) in Rab7a for Ser(140) in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.
Nencki Institute of Experimental Biology, Warszawa, Poland.
Magdalena
M
Osińska
Jolanta
J
Wiejak
Emilia
E
Wypych
Henryk
H
Bilski
Rafał
R
Bartosiewicz
Elżbieta
E
Wyroba
D000143:Acids / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000913:Antibodies, Protozoan / Q000378:metabolism; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D011992:Endosomes / Q000378:metabolism; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D006031:Glycosylation; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010247:Paramecium / Q000235:genetics / Q000378:metabolism*; D010588:Phagosomes / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D000143:Acids; D000913:Antibodies, Protozoan; D026901:Membrane Transport Proteins; D020543:Proteome; D000090802:rab7 GTP-Binding Proteins; D025262:Vacuolar Proton-Translocating ATPases; D020691:rab GTP-Binding Proteins