Distinct Membrane Domains on Endosomes in the Recycling Pathway Visualized by Multicolor Imaging of Rab4, Rab5, and Rab11

Abstract

Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

Sdmg1 is a component of secretory granules in mouse secretory exocrine tissues

“…Lastly, the subdomains may be an in vitro artifact generated during the purification or fixation of the secretory granules. Subdomains of SNAREs and lipids within membranes have been reported in various organelles where they have been suggested to be involved in sorting membranes and cargo to different destinations, or in clustering similar proteins together to facilitate protein-protein interactions between membranes (Sonnichsen et al, 2000;Lang et al, 2001). Our data suggesting that secretory granule membranes may also contain discrete subdomains is, therefore, consistent with observations in other membrane trafficking systems.…”

Intracellular trafficking of adeno-associated viral vectors

“…40,58,59,62 Transferrin trafficking has been extensively studied and shown to move through the early endosome to the PNRE. 76,77 The recycling of transferrin through the PNRE requires the coordinated interactions of several small GTPases (Rab5, Rab4, and Rab11) that direct the movement and fusion of early endosomes to the PNRE compartment. 77 The colocalization of CPV and AAV2 with transferrin has suggested the potential involvement of the PNRE in the processing of these viruses.…”

“…76,77 The recycling of transferrin through the PNRE requires the coordinated interactions of several small GTPases (Rab5, Rab4, and Rab11) that direct the movement and fusion of early endosomes to the PNRE compartment. 77 The colocalization of CPV and AAV2 with transferrin has suggested the potential involvement of the PNRE in the processing of these viruses. Additionally, we have observed significant overlap with Cy3-labeled AAV2 with the Rab11 PNRE compartment in certain cell types (unpublished data).…”

Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling

“…Thus, the rapid recycling of the interleukin-8 receptors CXCR1 and CXCR2 2 -adrenoceptors are inhibited by cytochalasin D, an actin filament-disrupting drug [8,9]. Rab4 has also a role in the recycling of internalized cargo to the cell surface (tra 2 -adrenoceptor) [10,11]. Whether this general model applies to the BK B 2 R has been tested by exploiting, along with cytochalasin D, ligands of microtubules that act in complementary manner:…”