PB1 and UBA domains of p62 are essential for aggresome-like induced structure formation

https://doi.org/10.1016/j.bbrc.2018.06.153Get rights and content
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Highlights

  • P62’s PB1 and UBA domains are required for the induction of ALIS.

  • YFP-tagged p62 is not a very mobile protein in ALIS.

  • P62 binds preferentially to K63-linked ubiquitin chains.

Abstract

ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62—PB1 and UBA—are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST–tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.

Keywords

ALIS
PB1
UBA
p62
FRAP
Ubiquitin

Abbreviations

ALIS
aggresome-like induced structures
DALIS
dendritic cell aggresome-like induced structures
DRiPs
defective ribosomal products
GFP
green fluorescent protein
GST
glutathione S-transferase
FRAP
fluorescence recovery after photobleaching
LPS
lipopolysaccharide
PB1
Phox and Bem1
PEI
polyethylenimine
UBA
Ubiquitin association
YFP
yellow fluorescent protein

Cited by (0)

1

Present Address: The Duke Human Vaccine Institute, Duke University, Durham, NC 27708.

2

Present Address: Division of Biology and Medicine, Brown University, Providence, RI 02912.