In the present study, we report that endogenous SMAP2 localizes mostly in REs and is essential for the retrograde transport of CTxB from REs to the Golgi. SMAP2 binds evection-2 and the RE localization of SMAP2 is abolished in cells depleted of evection-2. These findings suggest that evection-2 recruits SMAP2 to REs, thereby regulating the retrograde transport of CTxB from REs to the Golgi. We previously identified evection-2 as an RE protein that is essential for retrograde transport from REs to the Golgi. In the present study, we identified a second protein SMAP2, an Arf GAP, which functions in the retrograde transport of CTxB as evection-2. Furthermore, we found that evection-2 binds SMAP2 and is essential for the SMAP2 localization to REs. Although SMAP2 localized in REs, its distribution over the REs was not even. SMAP2 showed a high degree of co-localization with evection-2 and CTxB, but a lesser degree of co-localization with TfnR. As shown by the blue bar in a fluorescence intensity line scan profile, there was a region where TfnR did not co-localize at all with SMAP2. These observations suggested the two functionally separable subdomains in REs: one is a domain in which evection-2 and SMAP2 localize, functioning in the retrograde transport from REs to the Golgi and the other is a domain in which TfnR localizes, functioning the recycling transport from REs to the PM. In polarized cells, the PM contains distinct apical and basolateral domains. The REs in polarized cells function to maintain the PM integrity by recycling membrane proteins back to the correct PM domains. It is reported that the REs in polarized cells have subdomains where apical and basolateral cargoes are segregated. Our study suggests that REs has also subdomains in non-polarized cells. Therefore, the existence of subdomains in REs may be a general feature of RE membranes, which facilitates the correct sorting of different cargo proteins. Three Arf GAPs, besides SMAP2, are known to localize in endosomes or function in endosomal membrane transport. ACAP1 localizes in REs and is essential for the recycling pathway from REs to the PM through the recognition of specific sequences in recycled cargoes, such as TfnR. ACAP2 localizes to REs in PC12 cells stimulated with nerve growth factor, and regulates the neurite outgrowth. AGAP2 is required for the exit of Shiga toxin B subunit from EEs in HeLa cells. Combined with the previous findings, the current study postulates that a network of endosomal pathways into or out of REs can be regulated by a network of endosomal Arf GAPs. A 
recent study in BSC-1 cell has shown that the retrograde transport of STxB proceeds from PM to EEs to REs then to the Golgi. Thus, the retrograde transport of STxB from EEs to the Golgi may be dissected into two steps by two Arf GAPs. One is an AGAP2dependent pathway, which corresponds to EEs to REs. The other is a SMAP2-dependent pathway, which corresponds to REs to the Golgi.