Demonstration and characterization of the heterodimerization of ZnT5 and ZnT6 in the early secretory pathway.
The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.
Fukunaka
Ayako
A
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Suzuki
Tomoyuki
T
Kurokawa
Yayoi
Y
Yamazaki
Tomohiro
T
Fujiwara
Naoko
N
Ishihara
Kaori
K
Migaki
Hitoshi
H
Okumura
Katsuzumi
K
Masuda
Seiji
S
Yamaguchi-Iwai
Yuko
Y
Nagao
Masaya
M
Kambe
Taiho
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D055571:Secretory Pathway; D016415:Sequence Alignment
D027682:Cation Transport Proteins; C457225:SLC30A5 protein, human; C504822:SLC30A6 protein, human