The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular
matter have led to debate about contents and functional properties of exosomes. Here,
we employ high-resolution density gradient fractionation and direct immunoaffinity
capture to precisely characterize the RNA, DNA, and protein constituents of exosomes
and other non-vesicle material. Extracellular RNA, RNA-binding proteins and other
cellular proteins are differentially expressed in exosomes and non-vesicle compartments.
Argonaute 1–4, glycolytic enzymes and cytoskeletal proteins are absent from exosomes.
We identify Annexin A1 as a specific marker for microvesicles that are shed directly
from the plasma membrane. We further show that small extracellular vesicles are not
vehicles of active DNA release. Instead, we propose a new model for active secretion
of extracellular DNA through an autophagy- and multivesicular endosome-dependent,
but exosome-independent mechanism. This study demonstrates the need for a reassessment
of exosome composition and offers a framework for a clearer understanding of extracellular
vesicle heterogeneity. A reassessment of exosome composition establishes the differential
distribution of protein, RNA, and DNA between small extracellular vesicles and non-vesicular
extracellular matter and establishes that small extracellular vesicles are not vehicles
of active DNA release.