Rab22a affects the morphology and function of the endocytic pathway.
Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.
Mesa
R
R
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina.
Salomón
C
C
Roggero
M
M
Stahl
P D
PD
Mayorga
L S
LS
D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D006224:Cricetinae; D004285:Dogs; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C086223:rab22 protein, Canis familiaris; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D020691:rab GTP-Binding Proteins