Abstract
Macropinocytosis is an efficient process for the uptake of nutrients and solute macromolecules into cells from the external environment. Macropinosomes, which are surrounded by actin, are formed from the cell surface membrane ruffles and migrate toward the cell center. We have cloned the entire coding sequence of a member of the Rab family small GTPases, Rah/Rab34. It lacked a consensus sequence for GTP-binding/GTPase domain. Although wild-type Rah exhibited extremely low GTPase activity in vitro, it exerted appreciable GTPase activity in vivo. In fibroblasts, Rah was colocalized with actin to the membrane ruffles and membranes of relatively large vesicles adjacent to the ruffles. These vesicles were identified as macropinosomes on the basis of several criteria. Rah and Rab5 coexisted in some, but not all, macropinosomes. Rah was predominantly associated with nascent macropinosomes, whereas Rab5 was present in endosomes at later stages. The number of macropinosomes in the cells overexpressing Rah increased about 2-fold. The formation of macropinosomes by the treatment of platelet-derived growth factor or phorbol ester was also facilitated by Rah but suppressed by a dominant-negative Rah. Rah-promoted macropinosome formation was retarded by dominant-negative mutants of Rac1 and WAVE2, which are essential for membrane ruffling. These results imply that Rah is required for efficient macropinosome formation from the membrane ruffles.
Highlights
Introduction of Ras orRac1 induces membrane ruffling and macropinocytosis [13, 14]
Because Rah was most similar to Rab family proteins among small GTPase families and its amino acid sequence was identical to that of mouse Rab34, we compared its amino acid sequence with those of several other Rab family proteins (Fig. 1A)
Expression of the dominant-negative Rah highly reduced the number of actinassociated macropinosomes per cell both in the platelet-derived growth factor (PDGF)- and the phorbol 12-myristate 13acetate (PMA)-treated cells, but the levels remained higher than the control level (Fig. 7B)
Summary
Introduction of Ras orRac1 induces membrane ruffling and macropinocytosis [13, 14]. WAVE2 activated by IRSp53, a target protein of Rac1, is responsible for the Arp2/3 complexmediated formation of the branched actin filament meshwork in membrane ruffles (16 –18, 20). The dominantnegative mutant Rac1(T17N) interferes with membrane ruffling and macropinosome formation induced by growth factors, PMA, Ras, or Tiam1 [13, 15]. A mutant Rab5 protein defective in GTP-binding ability retards fluidphase pinocytosis in addition to receptor-mediated endocytosis [25], there has been no report indicating that any Rab family proteins are located to the membrane ruffles or macropinosomes and play direct roles in macropinosome formation.
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