RAB-GTPases act as master regulators in the endocytic and secretory pathways [55]. RAB23 is known to localize to the plasma membrane and the endocytic pathway [17]. However, multiple endocytic routes exist and the function of RAB23 in the context of membrane trafficking is largely unknown. Our data suggest that RAB23 functions in the clathrin-dependent route where RAB23 may participate in AP-2/clathrin-coated nascent vesicle formation at the plasma membrane (Fig. 1). Clathrin-coated nascent vesicle formation is an upstream event of early endosome which starts with AP-2 mediated cargo recognition, followed by clathrin coat assembly and subsequently vesicle scission [28, 30]. These nascent vesicles then start docking and fuse with EEA1-positive early endosomes [56]. Our study reveals that during clathrin-coated nascent vesicle biogenesis at the plasma membrane, RAB23 may function at multiple steps and thus, deficiency of RAB23 affects vesicle formation, internalization, transport and cell signaling. By hypergeometric analysis of microarray data obtained from differentially expressed genes in WT and RAB23 deficient mouse primary cells, we provide further evidence that RAB23 is involved in vesicle formation, endocytosis and cell signaling.
Vesicle transport keeps cargo identity intact by forming membrane-bound structures, and at the same time, it is essential to ferry the cargo from one cellular compartment to the target destination [57, 58]. Vesicle coats consist of an inner adaptor protein layer that recognizes and interacts with the cargo and G proteins, and a cage-like outer layer that wraps the adaptor layer [59, 60]. Upon binding with cargo and G proteins, adaptor proteins form a “prebudding complex”, the inner layer of vesicle. Subsequently, coat proteins are recruited to the prebudding complex to form a cage-like structure, known as the second layer of the vesicle. Finally, a GTPase-mediated hydrolysis, for instance driven by a RAB protein, detachs the nascent vesicle from the cell membrane [28]. Several other proteins including kinases are also involved in this process [12, 61]. Here, we show that in the clathrin-coated endocytic vesicle formation RAB23 interacts with β-adaptin subunit of the adaptor protein 2 (AP-2) complex but not with α-adaptin 1 and 2 (Fig. 1). There might be several reasons for this discrepancy. Firstly, the heterotetrametric AP-2 adaptor, which is a big complex of subunits; α-adaptin (⁓110 kDa), β-adaptin (⁓110 kDa), µ2-subunit (⁓50 kDa) and σ2 (⁓17 kDa) might be too big protein (⁓300 kDa) for a small vesicle protein antibody like RAB23 (⁓30 kDa) to pull down. α and µ2 are among the two subunits of AP-2 that are membrane bound and they might have interactions with many other proteins [62]. Even though RAB23 might have an interaction with these subunits through β-adaptin the only strong interaction subunit (β-adaptin) might come across with RAB23. Secondly, how the subunits of heterotetrameric AP-2 adaptor complex are assembled and dissemble might be another issue and the conformational changes which are highly dynamic. RAB23 might interact with β-adaptin in a conformational state where the complex might loosely become interconnected within themselves and may show only interaction with β-adaptin before their compact assembly or when they are loosely interconnected during conformational changes. In addition to β-adaptin, our result further demonstrates that RAB23 interacts with the clathrin assembly protein PICALM, BAR domain-containing protein endophilin A2 and vesicle scission protein cortactin (Fig. 1–3). This collectively suggests that the small GTPase protein RAB23 might be involved in multiple steps during clathrin-coated nascent vesicle formation.
Clathrin-mediated endocytosis is involved in several important cellular processes, including cargo sorting to the endosome at the plasma membrane and trans-Golgi network-mediated secretion of proteins [63, 64]. Perturbation of clathrin in multicellular organisms causes lethality [65, 66]. Also, genetically removing the clathrin-dependent core adaptor protein AP-2 results in embryonic lethality in worms, flies and mice [29, 67, 68]. And in the absence of AP-2, the endocytic patch at the plasma membrane takes a significantly longer time to produce vesicles, many patches are unable to form vesicles, retake cargo at the cell membrane and some patches are stacked at the membrane [69]. Similar to AP-2 disruption, we show that RAB23-deficient cells exhibit reduced transferrin internalization, retention of transferrin at the cell surface and spend longer time at the cell periphery, and some patches retake to the cell membrane as shown by time-lapse live cell imaging (Fig. 4, Videos 1–4). We demonstrate that transferrin patches are retained in the first step of nascent endosome marked by β-adaptin subunit of AP-2 and take a longer time to become endocytosed (Fig. 4). Furthermore, we show that the dynamics of co-localization between β-adaptin of AP-2 and clathrin are aberrant in RAB23 deficient cells (Fig. 5). These findings suggest that RAB23, AP-2 and clathrin functions together during endocytic patch formation and efficient patch internalization. Clathrin-mediated transferrin uptake also has been shown affected by several of kinases (92) when they are knocked down, which is an indication that clathrin-mediated endocytosis utilizes numerous proteins for efficient cargo internalization [61]. We demonstrate that RAB23 deficiency affected the number of vesicle formations with aberrant morphology upon BMP2 stimulation and altered BMP2 signaling. A previous study showed that endocytic clathrin adaptor PICALM directs endocytosis of R-SNARE (VAMP2, 3 and 8) [49]. Our study showed that PICALM interacts with RAB23 (Fig. 3) and that deficiency of RAB23 reduced the expression of VAMP8 (Fig. S11). We also show that deficiency of RAB23 causes reduced protein interaction between clathrin and AP-2 (Fig. 6). This abnormality of ligand-receptor endocytosis may not be restricted to transferrin, R-SNARE or BMP2 signaling through BMP receptors that we have shown in this study, we speculate that a common mechanism for several growth factors signaling pathways including Hedgehog signaling through ciliary vesicle formation, FGF and TGFβR signaling. RAB23 deficiency could alter many cellular signals that pass through the AP-2/clathrin route.
The clathrin route allows selective internalization of various metabolites carrying receptors [70, 71]. In RAB23-deficient mice, which mimic Carpenter syndrome in humans, the lack of functional RAB23 results in overt FGF10, Hh and Nodal signaling with consequent misexpression of downstream signal transducers (pERK1/2, Gli1, Lefty1/2 and Pitx2). This leads to overt osteogenesis at the growing bone ends in the developing skull, defective dorsal cell type specification during neural tube closure and abnormal left-right patterning of the heart [2, 22, 24, 72]. We have shown that RAB23 regulates musculoskeletal development through TGFβR and BMPR signaling [23]. These phenotypes go hand in hand with our findings that RAB23 modulates adaptor protein-mediated assembly of clathrin during the early steps of endocytosis, which has been shown to regulate growth factor-receptor signaling [29, 73, 74].
In summary, here we show a role for RAB23 in clathrin-mediated nascent vesicle formation and endocytosis. Our results show that in the endocytic pathway, RAB23 co-localizes with early endosomal markers EEA1 and RAB5. RAB23 also co-localizes with late endosomal marker RAB7 and autophagy marker LC3 A/B, reminiscing the previous finding that RAB23 is involved in autophagosome formation during group A streptococcus infection [75] Our results show, RAB23 interacts with adaptor proteins AP-2 and participates in the clathrin-coated vesicle formation. Clathrin-coated vesicle formation is a multi-step process that includes prebudding, clathrin assembly, curvature formation, and detachment of the nascent vesicle. RAB23 interaction with AP-2, PICALM, endophilin A2 and cortactin as well as co-localization with clathrin is required for proper vesicle formation and subsequent cargo internalization as well as cell signaling (Fig. 9). Our data suggest mechanistic insights into the cellular membrane trafficking functions of RAB23 in mammalian cells and shows that RAB23 may play a role at multiple steps during clathrin-coated nascent vesicle formation and endocytosis.