Elsevier

Blood Cells, Molecules, and Diseases

Volume 35, Issue 3, November–December 2005, Pages 398-403
Blood Cells, Molecules, and Diseases

Exosomes contain ubiquitinated proteins

https://doi.org/10.1016/j.bcmd.2005.08.005Get rights and content

Abstract

Multivesicular bodies (MVB) are endosomal compartments that contain multiple vesicles, which derive from a delimiting membrane by inward budding. Incorporation of membrane proteins into the luminal vesicles requires, at least for some model proteins, monoubiquitination of their cytoplasmic domain. The ubiquitin tags are recognized by a sorting machinery, of which some components are also monoubiquitinated. The ubiquitin tags and the sorting machinery are both removed before the vesicles bud into the MVB lumen. MVB vesicles are therefore not expected to contain monoubiquitinated proteins. The MVB content is degraded upon fusion of MVB with lysosomes. In many cell types, however, MVB can also fuse with the plasma membrane, resulting in secretion of their luminal vesicles into the extracellular milieu. Such secreted vesicles are termed exosomes, and their protein composition should, due to their origin, be identical to that of MVB luminal vesicles. We here demonstrate that exosomes contain polyubiquitinated proteins, many of which are not integrated into the membrane and relatively enriched as compared to total cell lysates. These results suggest that a subset of polyubiquitinated cytoplasmic proteins is incorporated into the MVB pathway. The potential cell biological relevance of this observation is discussed. Furthermore, these data indicate that ubiquitinated proteins can serve as markers for exosomes.

Introduction

Endosomal compartments receive proteins via the endocytic and the biosynthetic pathways and distribute them to distinct destinations, including the trans-Golgi network, the plasma membrane (domains) and lysosomes. The endocytic pathway comprises early sorting endosomes, recycling endosomes, late endosomes and lysosomes. Early sorting endosomes are the major entry site for endocytosed material. Late endosomes derive from early endosomes through a maturation process that involves a gradual change in contents and access for plasma-membrane-derived and TGN-derived transport vesicles [1]. Multivesicular bodies (MVB) have been described by electron microscopic studies already in the 1950s and 1960s, but it was not until the early 1980s that they were recognized as an intrinsic component of the endocytic tract [2], [3]. The formation of MVB is initiated at the early endosomal state as a result of the inward budding of the endosomal delimiting membrane. During the maturation of early endosomes to late endosomes, tens or even hundreds of 60–80 nm vesicles accumulate in their lumen [1], [4], hence, the name MVB. Only recently, it was demonstrated that the MVB luminal vesicles are truly free vesicles that have completely dissociated from the endosomal delimiting membrane [4]. Proteins that are not incorporated and thus retained at the MVB limiting membrane are either recycled to the TGN and plasma membrane or are delivered to the limiting membrane of lysosomes. The MVB luminal vesicles potentially have three distinct fates. First, their earliest acknowledged function is to target incorporated proteins to lysosomes for degradation, a process that requires either direct fusion of MVB with lysosomes [5], [6] or a poorly understood complex maturation process. Many membrane proteins employ this pathway for their degradation. For example, several growth factor receptors [7], e.g. the epidermal growth factor receptor [8], [9], are sorted at MVB for proteolytic degradation after ligand-induced endocytosis. Second, MVB can also serve as temporal storage compartments; recently, we demonstrated that immature dendritic cells (DC) store major histocompatibility complex (MHC) class II at MVB luminal vesicles [10]. Upon DC activation, the luminal vesicles fuse with the MVB-limiting membrane, from where subsequent transfer of MHC class II to the plasma membrane can proceed. The third possible fate of MVB occurs when their limiting membrane fuses with the plasma membrane. This process results in secretion of the MVB luminal vesicles, which are now termed exosomes [11], [12]. This pathway was first discovered in maturing reticulocytes [13], [14] and observed later for a large group of different cell types, including B lymphocytes, DC, T cells, mast cells, platelets and epithelial cells. The physiological role of exosomes is still unclear, but it is likely that they function in intercellular communication. Particularly, exosomes from DC have drawn lots of attention. These bear functional MHC class I– and class II–peptide complexes and have been shown to induce activation of specific T cells in vitro and in vivo [12], [15], [16], [17]. Similarly, exosomes derived from human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses in vitro [18]. Together, these data suggest a physiological role for exosomes in antigen presentation.

Incorporation of membrane proteins into the luminal vesicles of MVB is a selective and regulated process; monoubiquitination of the cytoplasmic domain of membrane proteins can serve as a signal for their sorting at MVB [2], [3], [19]. The formation of MVB luminal vesicles and the sorting of cargo protein therein depend on the function of at least 18 conserved proteins that were originally identified in the yeast Saccharomyces cerevisiae [3], [19]. Vps27/HRS contains two Ubiquitin Interaction Motifs (UIM) and is thought to act as an adaptor protein that binds both monoubiquitinated transmembrane proteins and clathrin and recruits monoubiquitinated membrane proteins to characteristic flat clathrin lattices on endosomal vacuoles [20], [21]. Vps27/HRS also interacts with ESCRT I (Endosomal Sorting Complex Required for Transport), a cytosolic protein complex that is transiently recruited to the endosomal membrane and functions in the sorting of ubiquitin-tagged transmembrane proteins into the MVB pathway. Whether the flat clathrin coat is strictly required for the interaction with ESCRT-I has not yet been established. After the interaction with ESCRT-I, two other protein complexes, ESCRT-II and ESCRT-III, sequentially associate with ubiquitinated membrane proteins, resulting in further concentration of cargo proteins [22], [23]. After sorting has been completed, ESCRT-III recruits the deubiquitinating enzyme Doa4, which removes the ubiquitin tag from the cargo transmembrane proteins prior to their incorporation into newly forming MVB vesicles [24]. A multimeric AAA-type ATPase, Vps4, dissociates the ESCRT-III complex before the luminal vesicles are formed. Several proteins of the MVB sorting machinery, including TSG101, a subunit of ESCRT-I, and HRS, are, like the cargo, being monoubiquitinated. As both the ubiquitinated components of the sorting machinery and the ubiquitin tags of the cargo are removed before MVB luminal vesicles are pinched off from the delimiting membrane, monoubiquitinated proteins are not expected to accumulate in MVB vesicles. Since exosomes are secreted MVB vesicles, one would not expect them to contain monoubiquitinated proteins either. We report here, however, that exosomes do contain polyubiquitinated non-integral membrane proteins and speculate on their relevance.

Section snippets

Cells

RN, an EBV-transformed human B-cell line RN (HLA-DR15), was cultured as described [18]. D1, a mouse immature splenic dendritic cell line, was cultured in 35% conditioned medium from R1 cells as described [28]. To remove exogenous exosomes from the culture media of RN and D1 cells, both fetal calf serum and R1 culture supernatants were ultracentrifuged at 140,000 × g for 60 min prior to use.

Antibodies, SDS-PAGE and Western blotting

Mouse monoclonal anti-human MHC class II (CR3/43) was from DakoCytomation (Glostrup, Denmark), mouse

Exosomes contain ubiquitinated proteins

In this study, we used two cell types, D1, a growth factor dependent long-term mouse DC culture [28], and RN [18], a human B-cell line. Cell culture media were first cleared from non-exosomal cell debris by differential centrifugation steps up to 30 min at 10,000×g. Subsequently, exosomes were pelleted by centrifugation for 60 min at 70,000 × g [18]. In line with previous observations [17], [18], exosomes from both sources, like the cells that secrete them, contained MHC II (Fig. 1A). Within

Acknowledgments

This paper is based on a presentation at a Focused Workshop on “Exosomes: Biological Significance” sponsored by The Leukemia and Lymphoma Society (Montreal Canada, May 20–21, 2005). We thank Dr. Ricciardi-Castagnoli (Univ. of Milan, Italy) for providing D1 and R1 cells.

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