Figure 8.

Rough ER vesicles that undergo a Mg²⁺ -induced shift in Suc gradients possess both AtPex16p-42 and 52-kD polypeptides. Arabidopsis suspension cell (7 d) clarified homogenates (1,500g, 15 min supernatant of 2,000 psi pressure cell-ruptured cells) were prepared in homogenizing medium with 5 mm MgCl₂ (+Mg²⁺) or 2 mm EDTA (−Mg²⁺). Microsomes (200,000g, 1 h) were layered onto 15% to 45% (w/w) Suc gradients with 5 mm MgCl₂ or 2 mm EDTA and centrifuged at 125,000g (2.5 h) in a SW28.1 rotor. Proteins in the 1 mL fractions were DOC solubilized, TCA precipitated, applied to SDS gels (about 150 μg protein per well), electroblotted to Immobilon, and probed with anti-AtPex16p-42 IgGs (1:100, overnight) or anti-castor calnexin antiserum (1:5,000, 1 h) followed by chemiluminescence detection of polypeptides. A pellet (P) at the bottom of both gradient tubes was resuspended in homogenizing medium, proteins were DOC solubilized, TCA precipitated, and applied (150 μg protein) to the left-most well of SDS gels.