Ubiquitously expressed secretory carrier membrane proteins (SCAMPs) 1-4 mark different pathways and exhibit limited constitutive trafficking to and from the cell surface

Abstract

Secretory carrier membrane proteins (SCAMPs) 1-4 are ubiquitously expressed and are major components of the eukaryotic cell surface recycling system. We investigated whether different SCAMPs function along distinct pathways and whether they behave like itinerant cargoes or less mobile trafficking machinery. In NRK cells, we show by immunofluorescence microscopy that different SCAMPs are concentrated mostly adjacent to one another in the trans-Golgi network and endosomal recycling compartment. By immunoelectron microscopy, they were shown to be close neighbors on individual transferrin-containing endosomal elements and on the plasma membrane. Within the internal endosomal network, SCAMPs are located distal to rab5-containing endosomes, and the individual isoforms appear to mark pathways that diverge from the constitutive recycling route and that may be distinguished by different adaptors, especially AP-1 and AP-3. Based on comparisons of SCAMP localization with endocytosed transferrin as well as live imaging of GFP-SCAMP1, we show that SCAMPs are concentrated within the motile population of early and recycling endosomes; however, they are not detected in newly formed transferrin-containing endocytic vesicles or in vesicles recycling transferrin to the surface. Also, they are not detected in constitutive secretory carriers marked by VSV-G. Their minimal recycling to the surface is reflected by their inability to relocate to the plasma membrane upon inhibition of endocytosis. Thus SCAMPs exhibit limited exchange between the cell surface and internal recycling systems, but within each of these sites, they form a mosaic with individual isoforms marking distinct pathways and potentially functioning as trafficking machinery at sites of vesicle formation and fusion. A corollary of these findings is that early endosomes exist as a distinct SCAMP-containing compartment and are not formed de novo by fusion of endocytic vesicles.

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

“…In these cells, as well as in HEK-293 cells, SERT and SCAMP2 staining neither overlaps significantly with TfR-positive endosomes, nor with the Golgi marker TGN38. Our data are in general agreement with a recent study by Castle and Castle, in which SCAMP2 showed limited co-localization with TGN38 and only partial co-localization with TfR in NRK cells (26). SCAMPs are predominantly associated with recycling rather than degradation pathways, suggesting that through the interaction with SCAMP2, SERT might be targeted to a distinct recycling compartment.…”

“…SCAMP2 belongs to a family of proteins that are major components of the eukaryotic cell surface recycling system. SCAMPs are ubiquitously expressed and are found on secretory organelles involved in regulated exocytosis, on recycling vesicles that shuttle to and from the plasma membrane and also on synaptic vesicles (18,19,26,30). SCAMP2 directly interacts with SERT both in a heterologous expression system and in rat brain tissue.…”

“…Although SCAMPs were shown to localize at least to a certain extend to the plasma membrane (24, 25), we were unable to biotinylate SCAMP2 under the conditions used (data not shown). This may be because of the fact that either the potential extracellular domains are very small, rendering them inaccessible for the biotinylation reagent, or because exposure of SCAMP2 to the cell surface is limited and/or transient (26,27). The reduction in SERT levels on the cell surface corresponds well with the changes in SERT uptake activity, suggesting that SCAMP2 exerts its effect by causing a redistribution of SERT.…”

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

“…SCAMPS are found in both the TGN and the endosomal recycling compartment in NRK cells, and they appear to be concentrated within the motile population of early and recycling endosomes . Thus, SCAMPs appear to be reliable indicators for postGolgi endocytic and exocytic trafficking in animal cells (FernandezChacon and Sudhof, 2000;Castle and Castle, 2005;Liu et al, 2005).…”

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

“…SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33,34) and have been shown to play a role in exocytosis (35)(36)(37)(38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals.…”