SCAMP 3 is a novel regulator of endosomal morphology and composition

Abstract

Secretory Carrier Membrane Proteins (SCAMPs) are transmembrane proteins that function in the plasma membrane, endosomes, and trans-Golgi network. Here we show that SCAMP 3 is a novel regulator of endosomal morphology and composition. Under certain nutrient-starved conditions, SCAMP 3 concentrates in enlarged early endosomes. The enlarged contain ubiquitylated and non-ubiquitylated SCAMP 3 as well as other SCAMPs, EEA1, and the ESCRT-0 protein Hrs. We demonstrate that SCAMP 3 is sufficient to recruit Hrs to the enlarged endosomes. Taken together, our results suggest a novel role for SCAMP 3 in modifying endosome structure through interactions that involve its ubiquitylation and ESCRT proteins.

Introduction

Endosomes are intermediate compartments that function between the plasma membrane, trans-Golgi Network (TGN), and lysosomes. They perform a myriad of functions including sorting and recycling endocytosed receptors, delivering cargo to the lysosome, processing pro-enzymes for activation, and sequestering signaling complexes [1], [2], [3]. These functions are central to homeostasis and cellular responses to changes in the environment.

Subtypes of endosomes are classified according to morphology, when they receive cargo, and the presence of various markers. Early endosomes receive cargo from the plasma membrane and TGN, contain markers such as EEA1 and Rab5, and have a tubulovesicular morphology with some intraluminal vesicles [2], [4]. Cargo in the early endosomes can be directed to late endosomes or recycling endosomes, or can be recycled directly back to the plasma membrane. The recycling endosome is a network of tubulovesicular compartments and is often identified by the presence of Rab11 [2], [5]. Late endosomes are spherical and contain intraluminal vesicles; thus, they are also known as multivesicular bodies (MVBs). They are thought to originate from early endosomes, contain markers such as Rab7, and deliver cargo to the lysosome [2], [6], [7].

The structure and function of each type of endosome is determined by its protein composition. Many of these proteins function as adaptor molecules to help sort cargo to their target destination. At the early endosomes, membrane proteins that are targeted for degradation are sorted into intraluminal vesicles by the endosomal sorting complexes required for transport (ESCRTs). This cargo sorting often involves the covalent attachment of ubiquitin, a small 76 aa protein, as well as additional accessory proteins [8]. Overexpression or depletion of ESCRT proteins is known to affect endosome morphology and in many cases may affect the trafficking of one or more cargo molecules through the compartment [9], [10], [11], [12], [13].

Secretory Carrier Membrane Proteins (SCAMPs) are abundant transmembrane proteins found in endosomes, as well as the plasma membrane and TGN [14], [15]. SCAMP homologs have been identified in metazoans, plants, and some fungi. In humans, the SCAMP family consists of three long isoforms, SCAMPs 1–3, and two shorter isoforms, SCAMPs 4 and 5. SCAMPs are known to affect intracellular trafficking of various transporters and receptors [16], [17], [18] and are thought to interact with proteins involved in protein sorting and vesicle formation [19], [20]. In particular, SCAMP 3 has been shown to modulate degradation of the epidermal growth factor receptor (EGFR) via interaction with ESCRT proteins and ubiquitylation [19], [21].

In this study we present evidence that SCAMP 3 is a novel regulator of endosomal morphology and composition. We demonstrate that under specific serum-starved conditions, SCAMP 3 regulates the size of early endosomes and is sufficient for recruiting the ESCRT-0 protein Hrs to early endosomes. Under these conditions SCAMP 3 is also highly ubiquitylated. These results suggest a potential mechanism by which cells can modify endosome functions through ubiquitylation of SCAMP 3.