Thermolysin Is a Suitable Protease for Probing the Surface of Intact Pea Chloroplasts

Abstract

ABSTRACISeveral proteases, ie., pronase, a mixture oftrypsin and chymotrypsin, and thermolysin were screened as potential surface probes of isolated intact pea (Pisum satiium var Laxton's Progress No. 9) chloroplasts. Of these, only thermolysin met the criteria of a suitable probe. Thermolysin destroyed outer envelope polypeptides, but did not affect inner envelope polypeptides, envelope permeability properties or such chloroplast activities as metabolite transport and 02 evolution.Methods involving protease treatment have been extensively used for probing the surfaces of cells, organelles, and isolated membranes (5, 13). In general, the exogenously added protease is able to digest proteins exposed on the outer surface of such structures but is prevented from gaining access to proteins on the other side ofthe membrane barrier. Thus, protease treatment has been used for a variety of purposes including: localization of membrane proteins and enzymes (5, 13), determining the orientation of transbilayer polypeptides (5, 13), removing proteins adsorbed or bound to the surface of membrane-bounded structures, etc. (12).The usefulness of protease treatment for such purposes relies on the inability of the protease to penetrate the bounding membrane. In many cases membrane rupture leading to protease penetration is not a problem because the debris from the ruptured material can be excluded from subsequent analysis by repurifying the remaining intact cells, organelles, etc. However, for double membrane-bounded organelles (chloroplasts, mitochondria), such a repurification procedure is not adequate since rupture of the outer membrane is not necessarily accompanied by rupture of the organelle. Consequently, special precautions must be undertaken to demonstrate that the exogenous proteases are limited in effect to the outer membrane.Recently, several studies including one from our own laboratory have utilized proteases as a surface probe of chloroplasts (3,4,6,8,12). Due to the complexity of the chloroplast-bounding membrane, the envelope, and because of certain inconsistencies we found in our own prior experiments using protease treatment (4), we have examined several proteases to determine their usefulness as surface probes of pea chloroplasts. This communication documents the use of thermolysin, a relatively nonspecific metalloprotease, as a probe whose effects are limited to the outer envelope membrane of isolated, intact pea chloroplasts. MATERIALS AND METHODSProteases were obtained from Sigma; radiochemicals were obtained from New England Nuclear. All other chemicals were reagent grade.Intact chloroplasts were purified from homogenates of 2-to 3-week-old pea seedlings (Pisum sativum var Laxton's Progress No. 9) by differential centrifugation followed by Percoll density gradient centrifugation as previously described (4). Chloroplast envelope membranes were isolated and subsequently subfractionated into inner and outer membrane fractions by the procedure described previously (4).For thermolysin treatment, purified intact...

A guanosine 5?-triphosphate-dependent protein kinase is localized in the outer envelope membrane of pea chloroplasts

“…The results of SDS-PAGE of purified envelope membranes are shown in Fig. 1 and demonstrate the purity of the respective membrane population and the typical distribution of envelope marker proteins (Cline et al 1984). As can be seen in lanes 1-5, when [7-32p]ATP is used as the phosphoryl donor, each of the chloroplast fractions yields a distinctive pattern of labeled polypeptides.…”

“…1 ; Keegstra and Yousif 1986). Chloroplasts were protease-treated with thermolysin at 200 gg protease, mg ~ chlorophyll (Joyard et al 1983;Cline et al 1984) and separated inner and outer envelope membranes were purified from these plastids (Keegstra and Yousif 1986). Soluble chloroplast proteins were isolated after hypotonic lysis of purified intact chloroplasts and subjected to centrifugation at 50000.g for 30 min.…”

Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts

“…One of these proteins had a size of around 86 kDa and was susceptible to thermolysin treatment (Cline et al, 1984;Joyard et a/., 1983). Antibodies which we have raised against the outer envelope protein of 86 kDa (OEP 86) recognized the high molecular weight component in fractions 15-1 8 of the sucrose gradient ( Figure 5).…”

The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids

“…Furthermore, it has been shown that protease treatment of intact chloroplasts diminishes the import, indicating the involvement of surface exposed proteins [7]. Much less attention has been given to the possible roles of envelope lipids.…”