Ct. Okamoto et Yy. Jeng, AN IMMUNOLOGICALLY DISTINCT BETA-ADAPTIN ON TUBULOVESICLES OF GASTRICOXYNTIC CELLS, American journal of physiology. Cell physiology, 44(5), 1998, pp. 1323-1329
Clathrin and the gamma-adaptin subunit of the AP-1 clathrin adaptor ha
ve been previously identified on H-K-ATPase-rich tubulovesicles from g
astric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y.
Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 (Cell Phy
siol. 43): C1017-C1029]. We further characterized this AP-1 adaptor fr
om rabbit and hog tubulovesicles biochemically and immunologically. Cl
athrin coat proteins were stripped from purified tubulovesicular membr
anes and fractionated by hydroxyapatite chromatography. The AP-1 adapt
or appears to elute at 200 mM sodium phosphate, based on the presence
of proteins in this fraction that are immunoreactive with antibodies a
gainst three of the four subunits of this heterotetrameric complex: th
e gamma-, mu(1)-, and sigma(1)-adaptin subunits. Although the putative
beta-adaptin subunit in this fraction is not immunoreactive with the
anti-beta-adaptin monoclonal antibody (MAb), this beta-adaptin is immu
noreactive with polyclonal antibodies (PAbs) directed against the pept
ide sequence -Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val(640), a regi
on conserved between beta(1)- and beta(2)-adaptins that is thought to
be involved in the binding of clathrin heavy chain. Immunoprecipitatio
n of the AP-1 adaptor complex from this fraction with anti-gamma-adapt
in MAb 100/3 resulted in the coimmunoprecipitation of the beta-adaptin
that did not react with the anti-beta-adaptin MAb but did react with
the anti-beta-adaptin PAbs. In contrast, immunoprecipitation of the AP
-1 adaptor complex from crude clathrin-coated vesicles from brain resu
lted in the coimmunoprecipitation of a beta-adaptin that was recognize
d by both the anti-beta-adaptin MAb and PAbs. These results suggest th
at the tubulovesicular AP-1 adaptor complex may be distinct from that
found in the trans-Golgi network and may contain an immunologically di
stinct beta-adaptin. This immunologically distinct beta-adaptin may be
diagnostic of apical tubulovesicular endosomes of epithelial cells.