Since we found both LC3 and appear to diffuse much slower than soluble Venus, this raised the possibility that the slower than expected diffusion may be the result of homo-oligomerization of LC3 or , or that multiple copies of LC3 and may be contained within the same multiprotein complex. To test this possibility we performed acceptor photobleaching FRET measurements on cells coexpressing Venus- and Cerulean-tagged versions of LC3 and . In the cytoplasm we found no evidence for the presence of LC3 or homo-oligomers. The absence of detectable FRET between LC3 and LC3, or between and , implies that if more than one copy of LC3 or is present in the same slowly diffusing complex, these molecules are positioned at distances greater than 10 nm from one another in the cytoplasm. However, due to the orientation requirements for FRET, the absence of energy transfer efficiency cannot rule out the possibility of homo-oligomerization, or more than one LC3 in a complex. For example, if two LC3’s are bound to as seen in the crystal structure, it is possible their N-terminal fluorescent protein labels are positioned on opposite sides of (), preventing a detectable amount of energy transfer from occurring.