Sec-independent protein translocation by the maize Hcf106 protein

Science

Volumn 278, Issue 5342, 1997, Pages 1467-1470

  Settles, A Mark ^a   Yonetani, Ann ^a,d   Baron, Aimee ^b   Bush, Daniel R ^a,e   Cline, Kenneth ^c   Martienssen, Rob ^a  

^a Howard Hughes Medical Institute Cold Spring Harbor Laboratory Cold Spring Harbor   (United States)

^b EMORY UNIVERSITY SCHOOL OF MEDICINE   (United States)

^c UNIVERSITY OF FLORIDA   (United States)

^d UNIVERSITY OF CALIFORNIA   (United States)

^e UNIVERSITY OF ILLINOIS AT URBANA CHAMPAIGN   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN; VEGETABLE PROTEIN;

ARTICLE; BACTERIAL GENE; CHLOROPLAST; CONTROLLED STUDY; MAIZE; NONHUMAN; PRIORITY JOURNAL; PROTEIN LOCALIZATION; PROTEIN TRANSPORT;

AMINO ACID SEQUENCE; BACTERIAL PROTEINS; CARRIER PROTEINS; CHLOROPLASTS; EVOLUTION, MOLECULAR; GENES, PLANT; HYDROGEN-ION CONCENTRATION; INTRACELLULAR MEMBRANES; MEMBRANE PROTEINS; METHYLAMINES; MOLECULAR SEQUENCE DATA; MUTATION; OPEN READING FRAMES; PLANT PROTEINS; SEQUENCE ALIGNMENT; ZEA MAYS;

EID: 0030722626     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5342.1467     Document Type: Article

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44. 3 (10 mM) on ice 10 min before the addition of diluted precursor. The chloroplasts were post-treated with thermolysin (0.1 mg/ml) on ice for 40 min and repurified on a 35% Percoll cushion with 5 mM EDTA. The radiolabeled proteins were separated by SDS-polyacrylamide gel electrophoresis (SOS-PAGE) and visualized with fluorography.
45. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
46. 2 and incubation on ice for 10 min. Thylakoids were purified by slow-speed centrifugation and washed once in lysis buffer. Supernatant proteins were concentrated by trichloracetic acid (TCA) precipitation. Membrane and supernatant pellets were resuspended in equal volumes of loading buffer, separated by SDS-PAGE, and blotted to nitrocellulose. The blot was incubated and stripped, and reprobed with several antibodies with the use of enhanced chemiluminescence.
47. Thylakoid membranes were prepared as described (15). Thylakoids were pelleted and resuspended (in 50 μg of chlorophyll per milliliter) in each chaotropic solution with 1 mM phenylmethylsulfonyl fluoride. The membranes were then incubated on ice for 30 min. Sonicated membranes were treated in a bath sonicator four times for 10 s each time. The extracted thylakoid membranes were separated by differential centrifugation. Supematants were concentrated by TCA precipitation. The samples were separated by SDS-PAGE, and protein gels were blotted with antibodies against HCF106, OE33 (13), and LHCP.
48. Purified chloroplasts were lysed, and thylakoids were separated by low-speed centrifugation. The thylakoids were then resuspended in lysis buffer or 1 % Triton X-100 (0.3 mg of chlorophyll per milliliter) and treated with thermolysin (0.1 mg/ml final concentration) for 30 min on ice. Protease digestion was stopped by addition of EGTA (50 mM final concentration), the samples were separated by SDS-PAGE, and protein gels were blotted with antibodies against HCF106 and OE33(13).
49. Supported by USDA grant 94-37304-1043 and NSF grant MCB-9220774 to R.M. and by NSF grant MCB-9419827 to K.C.C. We thank A. Barkan, R. Voelker, and B. Taylor for sharing reagents, seed, and their thoughts on hcf106; R. Henry for advice on chloroplast import assays; L. Das for technical assistance; and T. Mulligan for plant care.