Figure 4.

Protease Treatments of the Chloroplast Surface Reveal That the 1-MD Translocation Complex Is Resistant to Thermolysin but Partially Sensitive to Trypsin.

(A) to (E) The translocation intermediate was generated under 0.5-mM ATP as described in Methods. The pea chloroplast suspension was divided into six aliquots (lanes 1 to 6) and washed twice with HS buffer in the absence of protease inhibitor cocktail. Chloroplasts carrying [³⁵S]pSSU were treated with the indicated concentrations of thermolysin (Thl) or trypsin (Trp) for 20 min on ice. After inactivation of the proteases, the chloroplast pellet was solubilized and subjected to BN-PAGE ([A], top) and SDS-PAGE ([A], bottom). When using trypsin, trypsin inhibitor was added to BN-PAGE sample buffer.

(B) Aliquots of the same samples in (A) were subjected to SDS-PAGE and immunoblotting with antibodies against indicated proteins. Arrows indicate intact Toc159, 86- and 52-kD fragments of Toc159, and Tic22. An asterisk denotes a nonspecific cross-reacting band.

(C) A gel strip corresponding to lane 1 (without protease) of (A) was subjected to 2D-BN/SDS-PAGE.

(D) A gel strip corresponding to lane 3 (with thermolysin) of (A) was analyzed as in (C).

(E) A gel strip corresponding to lane 6 (with trypsin) of (A) was analyzed as in (C). The three excised first-dimension BN-PAGE gel strips used in (C) to (E) were derived from the same first-dimension BN-PAGE gel. The positions of the 1-MD translocation intermediate complex, a partially degraded translocation intermediate complex, free pSSU, unassembled mSSU, and dissociated SSU-DP are indicated by brackets. mSSU assembled into the 520-kD Rubisco holoenzyme is indicated by arrows ([C] to [E]).