To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma-membrane-associated proteins following treatment with 150mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt-sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two-phase partitioning, and plasma-membrane-associated proteins were separated by IEF/SDS-PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5-fold in response to salt stress. Of these proteins, nine were upregulated and nine were down-regulated. MS analysis indicated that most of these membraneassociated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine-rich-repeat type receptor-like protein kinase, OsRPK1, was identified as a salt-responding protein. Immuno-blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno-histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.

Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotindirected affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membraneassociated cytosolic proteins by harsh washes with highsalt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation as almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 íg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

前期研究表明，水稻根尖细胞质膜类受体蛋白激酶OsRLK的表达受盐胁迫诱导。为了进一步研究该激酶的生理功能，通过反转录PCR得到OsRLK胞外区cDNA片段，将其亚克隆至pET29a原核表达载体并在大肠杆菌中实现了高表达，表达量约为细胞总蛋白的30%。重组蛋白经SDS-PAGE分离，染色切胶收集后，作为抗原免疫新西兰家兔，分离抗血清，经纯化得到1：20000效价的多克隆抗体。Western blot结果显示，该抗体能特异识别在原核表达系统内表达的抗原，以及水稻根尖细胞质膜组分中的LRR型类受体蛋白激酶，并且在蛋白质水平证实该激酶为盐胁迫响应蛋白。

By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N7/C5 split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-speci®c cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 AÊ), N,N0-ophenylenedimaleimide (6 AÊ) and N,N0-p-phenylenedimaleimide (10 AÊ). Mutant Asp240→Cys (helix VII)/Lys319→Cys (helix X) cross-links most ef®ciently with the 3 AÊ reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H+ symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H+ from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).

It has been shown that mitochondria play a pivotal role in plant programmed cell death (PCD). Previous study established a salt stress-induced PCD model in rice (Oryza sativa L. cv. WYJ 8th) root tip cells, demonstrated by DNA laddering, cytochrome c release, and TUNEL positive reaction. In this study, the role of mitochondria during the early phase of PCD (2h-PCD) was analyzed in rice roots. After 2h-PCD induction, the integrity of mitochondria decreased slightly, consistent with a small release of cytochrome c. 2h-PCD partially inhibited electron transport, resulting in oxidative burst in mitochondria. However, ATP production maintained constant. Mitochondria proteome were analyzed by two-dimensional IEF/SDS-PAGE before and after 2h-PCD induction, and eight PCD-related proteins were identified. Among them, four proteins were up-regulated after PCD induction, which included glycoside hydrolase, mitochondrial heat shock protein 70, 20S proteasome subunit, and Cu/Zn-superoxide dismutase, and four were down-regulated, namely ATP synthase beta subunit, cytochrome c oxidase subunit 6b, S-adenosylmethionine synthetase 2, and transcription initiation factor eIF-3 epsilon. These results suggested that ATP synthase may not be the major producer of ATP in mitochondria during the early stage of PCD in rice. Glycoside hydrolase may be involved in ETC impairment and ROS burst, and mitochondrial HSP70 is a potential candidate for PCD regulation. The possible roles of other proteins on PCD initiation were also discussed.