Self-assembly is important for TIP47 function in mannose 6-phosphate receptor transport.
TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the trans-Golgi network in vitro and in living cells. TIP47 occurs in cytosol as an oligomer; it chromatographs with an apparent mass of approximately 300 kDa and displays an S-value of approximately 13. Recombinant TIP47 forms homo-oligomers that are likely to represent hexamers, as determined by chemical cross-linking. Removal of TIP47 residues 1-151 yields a protein that behaves as a monomer upon gel filtration, yet is fully capable of binding mannose 6-phosphate receptor cytoplasmic domains. The presence of an oligomerization domain in the N-terminus of TIP47 was confirmed by expression of N-terminal residues 1-133 or 1-257 in mammalian cells. Co-expression of full-length TIP47 with either of these fragments led to the formation of higher-order aggregates of wild-type TIP47. Furthermore, the N-terminal domains expressed alone also occurred as oligomers. These studies reveal an N-terminal oligomerization domain in TIP47, and show that oligomerization is not required for TIP47 recognition of mannose 6-phosphate receptors. However, oligomerization is required for TIP47 stimulation of mannose 6-phosphate receptor transport from endosomes to the trans-Golgi in vivo.
Sincock
Paul M
PM
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.
Ganley
Ian G
IG
Krise
Jeffrey P
JP
Diederichs
Sven
S
Sivars
Ulf
U
O'Connor
Brian
B
Ding
Li
L
Pfeffer
Suzanne R
SR
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D000070796:Perilipin-3; D011108:Polymers / Q000378:metabolism*; D011257:Pregnancy Proteins; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C038196:PLIN3 protein, human; D000070796:Perilipin-3; D011108:Polymers; D011257:Pregnancy Proteins; D017527:Receptor, IGF Type 2; D033921:Vesicular Transport Proteins