Figure 2.

The PD-Enriched Membrane Fraction Predominantly Contains PD-PM-Derived Vesicles and Is Not Contaminated by Other Membrane Compartments.

(A) Left, surface view electron micrograph of a purified wall fragment containing embedded PD revealing the concentric arrangement of the PD-PM (black arrowhead), the cytoplasmic sleeve, and the central desmotubule (black dashed arrow). Cellulose fibers are visible around PD (white arrows). Bar = 50 nm. Right, interpretative diagram of the structure of PD (longitudinal section and surface view) in purified walls as shown in the left panel. CW, cell wall.

(B) Immunoblot analysis showing that the PD fraction is enriched in PD-PM markers (PDLP1 and PDCB1) while deprived of markers from the ER (SMT1 and BiP), vacuole (V-ATPase), trans-Golgi network (TGN; echidna), Golgi (membrine11), thylakoid (P16), soluble proteins/stroma (Rubisco), and mitochondria (Nad9). The desmotubule-associated ER protein calreticulin is detected in the PD fraction but not enriched compared with the microsomal (μ) protein extract. The same amount of protein was loaded in each lane (7 μg).

(C) The protein profile of the PD-enriched membrane fraction is distinct from that of the PM and microsomal (μ) extracts. The same amount of protein was loaded in each lane (20 μg).

(D) Electron micrograph of the PD-enriched membrane fraction showing that the final extract is composed of membrane vesicles ∼50 to 150 nm in diameter. Note that internal desmotubule-like structures are also detected (arrow). Bars = 50 nm.

(E) to (H) Electron micrographs of PDCB1 (E) and PDLP1 (G) immunogold labeling of the PD fraction. The density of gold particles per vesicle was calculated ([F] and [H]) for both preimmune antisera (n = 218 vesicles for PDCB1; n = 97 vesicles for PDLP1 [not shown]) and specific antisera (n = 163 vesicles for PDCB1; n = 75 vesicles for PDLP1). Bars = 50 nm.