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* This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Research Grants (11B-1 and 14B-4) for Nervous and Mental Disorders from the Ministry of Health, Labor, and Welfare of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s)
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.
phorbol 12-myristate 13-acetate
guanine nucleotide exchange factor
platelet-derived growth factor
enhanced green fluorescent protein
rhodamine B isothiocyanate
4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
Endocytosis in eukaryotic cells serves to maintain cellular and organismal homeostasis by taking up fluids and macromolecules, signaling molecules, and their receptors from the external environment (
). There are at least five independent endocytic processes: clathrin-dependent endocytosis mediated by clathrin-coated vesicles (100–150 nm in diameter), caveolin-dependent endocytosis mediated by caveolae (50–80 nm), clathrin- and caveolin-independent endocytosis, macropinocytosis, and phagocytosis (
). Macropinosomes are not coated with clathrin or caveolin but surrounded by actin at early stages. Macropinocytosis provides an efficient process for non-selective uptake of nutrients and solute macromolecules. It also accounts for internalization of extracellular antigens by professional antigen-presenting cells like dendritic cells. Furthermore, macropinocytosis is postulated to play important roles in chemotaxis by regulating plasma membrane-actin cytoskeleton interaction and membrane trafficking. Some pathogenic bacteria such asSalmonella typhimurium and Shigella flexneri also exploit macropinocytosis to invade the cells (
Treatment of various types of cultured cells with growth factors, cytokines, phorbol esters such as phorbol 12-myristate 13-acetate (PMA),1 or diacylglycerol elicits rapid and dramatic membrane ruffling and macropinocytic responses (
Rab family small GTPases play essential roles in the endocytic and exocytic pathways. It is well established that Rab proteins function in the targeting and docking of the vesicles to their acceptor membranes. However, it has become evident that at least some of them exert their functions in multiple steps of vesicular trafficking, including vesicle formation, vesicle motility, membrane remodeling, and vesicle fusion as well as vesicle targeting and docking (
). In mammalian cells, >50 members of Rab family proteins have been identified. These proteins are associated with particular vesicle membrane compartments and function in specific stages of the diverse vesicle trafficking events. Among them, Rab5 is located to the membranes of clathrin-coated vesicles and early endosomes. It is involved in receptor-mediated endocytosis and fluid-phase pinocytosis (
). Introduction of a constitutively active Rab5 mutant in cells stimulates the rate of endocytosis and homotypic fusion of early endosomes, whereas its dominant-negative mutant prevents the vesicle fusion (
), we have cloned in the present study the cDNA containing the entire coding region of mouse Rah. Rah was colocalized with actin to the membrane ruffles and macropinosome membrane. During macropinosome biogenesis, Rah associated with nascent macropinosomes seemed to be replaced by Rab5 on the early endosomes. Overexpression of Rah elevated the number of macropinosomes, whereas the expression of a dominant-negative form of Rah prevented the formation of macropinosomes induced by platelet-derived growth factor (PDGF) or PMA. On the other hand, Rah-promoted macropinosome formation was retarded by dominant-negative mutants of Rac1 and WAVE2. Thus, Rah is likely to play crucial roles in the formation of macropinosomes from the membrane ruffles.
We have cloned the entire coding sequence of Rah/Rab34. It consists of 259 amino acids, and its large size is ascribed to its long N-terminal sequence. Usually the N- and C-terminal sequences are diverged even among the same family of small GTPases. Rah was most closely related to Rab36, the largest Rab family protein, over its entire length and in its N terminus. Although roles of the N-terminal sequences of small GTPases have not been well understood, some small GTPases bind particular proteins at their N termini. For instance, RalA interacts at its N terminus with phospholipase D1 in collaboration with Arf1 (
). Rnd1 and RhoE interfere with both Rho- and Rac-mediated reorganization of the actin cytoskeleton. Deletion of the N-terminal six amino acids in Rnd1, however, results in deprivation of the antagonistic effects on the cytoskeleton (
). Thus, the long N-terminal sequence of Rah seems to be involved in the interaction with some proteins to exert its cellular functions. The similarity between the N terminus of Rah and the corresponding sequence of Rab36 raises the possibility that these proteins share the proteins interacting with these sequences. Furthermore, Rah and Rab36 exhibit the identity between their core effector domains and close similarity between their extended effector domains. This may imply that these proteins also share several target proteins.
Although small GTPases have four conserved motifs for GTP/GDP-binding and GTPase activities, Rah lacked the fourth motif. In addition, the fourth motif was abrogated in Rab36 as well. The low intrinsic GTPase activity of Rah(wt) in vitro might be ascribable to the lack of this motif. However, Rah(wt) exhibited substantial GTPase activityin vivo comparable to the activity of Rab5(wt). In addition, even the constitutively active Rah(Q111L) and Rab5(Q79L) exerted appreciable GTPase activity in vivo. This is presumably due to the presence of specific GAP activities in cells as has been shown with Rab3A (
). Particularly, the GAP activity acting on Rah seems to be strong, because Rah(Q111L) showed higher GTPase activity than did Rab5(Q79L) and almost comparable to that of Rah(wt) in vivo. Furthermore, certain functions, including endocytic ability, are indistinguishable between Rab5(wt) and Rab5(Q79L) (
). This may explain why the phenotype of the cells transfected with Rah(wt) and that with Rah(Q111L) were indistinguishable.
Rah was colocalized with actin to the membrane ruffles and membranes of relatively large vesicles adjacent to the ruffles. In addition, time-lapse microscopy showed that the vesicles were actually formed from the membrane ruffles. These vesicles are identified as macropinosomes because of their large size, formation from the membrane ruffles, incorporation of dextran from the medium, and colocalization with actin. Although >50 members of Rab family proteins have been identified in mammalian cells (
), Rah is the first Rab family protein, to our knowledge, associated specifically with both the membrane ruffles and macropinosome membranes. Rab5 is located to the membranes of clathrin-coated vesicles and early endosomes. It participates in receptor-mediated endocytosis and fluid-phase pinocytosis (
). Coexpression of Rah and Rab5(Q79L) showed that Rah but not Rab5 was associated with membrane ruffles. Although Rah and Rab5 were colocated in some vesicles, the Rah-containing vesicles were generally present more peripherally in the cells, whereas the Rab5-containing vesicles occupied more central areas of the cells. These results suggest that, during macropinosome biogenesis, Rah acts at early stages and Rab5 functions at later stages and that Rah is replaced by Rab5 on macropinosomes.
The formation of membrane ruffles and macropinosomes are induced by the treatment of cells with growth factors or PMA (
). Alternatively, PI3K, a target protein of Ras, may link Ras with Rac1 through its lipid product phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). This lipid binds to and activates Vav and Sos, which serve as the GEFs for Rac1 (
). A presumable pathway for growth factor-induced membrane ruffling and macropinocytosis is summarized in Fig. 8.
We showed here that the expression of Rah(wt) and Rah(Q111L) facilitated the formation of macropinosomes over the control level. In contrast, the dominant-negative Rah(T66N) slightly retarded the macropinosome formation under the control level. When the cells were treated with PDGF or PMA, membrane ruffling and macropinosome formation were promoted. Expression of Rah(wt) in the PDGF- or PMA-treated cells further promoted the macropinosome formation. Expression of the dominant-negative Rah reduced the macropinosome formation, but the levels remained higher than the control level. On the other hand, the degree of membrane ruffling was not affected by any of these Rah proteins. These results indicate that Rah is concerned with macropinosome formation synergistically with growth factors (including serum growth factors in the growth medium) or PMA. Because Rah is not involved in membrane ruffling, however, Rah does not seem to constitute the signaling pathway activated by growth factors (Fig. 8).
We further showed that Rah-induced macropinosome formation was retarded to some degree by the dominant-negative H-Ras(S17N). On the other hand, Rac1(T17N) almost completely inhibited macropinosome formation, whereas the dominant-negative WAVE2(ΔV) suppressed it close to the control level. The only moderate retardation by H-Ras(S17N) might be ascribed to the presence in 10T1/2 cells of multiple endogenous Ras proteins (H-Ras, K-Ras, and N-Ras), which are structurally closely related but functionally distinctive (
). Thus, if GEFs specifically act on each member of Ras in vivoand H-Ras(S17N) sequester a particular GEF, H-Ras(S17N) may not efficiently interfere with macropinosome formation. Although WAVE2 is activated by Rac1-IRSp53 to induce membrane ruffling and subsequent macropinocytosis, there are two other WAVE isoforms, WAVE1 and 3 (
). Membrane ruffling induced by growth factors or PMA may result in spontaneous basal level macropinosome formation, which does not require the aid of Rah. Rah was colocalized with actin to the membrane ruffles and macropinosome membranes. Thus, the primary role of Rah seems to be the formation of macropinosomes by closing membrane ruffles through regulating actin reorganization. In this manner, Rah might be concerned with efficient macropinosome formation synergistically to the action of growth factors or PMA. This postulation is supported by the findings that Rah(T66N) is associated with membrane ruffles but not with macropinosomes and that both Rah and the actin coat disappear from macropinosomes as the vesicles migrate to the cell center. In this context, it should be noted that PI3K is not necessary for membrane ruffling but rather functions in the closure of membrane ruffles to form macropinosomes and phagosomes in macrophages (
). If this is also the case for fibroblasts, PI3K and PI(3,4,5)P3 may participate in the activation of Rah (Fig. 8). The closure of membrane ruffles is accompanied by a membrane fusion process. Some Rab family proteins, including Rab5, are involved in vesicle membrane fusion (
). Thus, it is of interest to examine whether Rah is required for the plasma membrane fusion at the ruffles. Identification of target proteins of Rah and their binding proteins may help to answer these questions.
We appreciate Drs. Chihiro Sasakawa and Toshihiko Suzuki for valuable discussions.