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Compartmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei

¹ Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
² Centro de Biología Molecular `SO', Lab CX-203, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain

View larger version (65K): [in a new window]   Fig. 2. Effects of PEX2 depletion on matrix protein import and glycosome morphology in bloodstream form trypanosomes. Cells were grown without Tet (control, -Tet) or in the presence of 100 ng/ml Tet for 24 hours (+Tet). For the top and middle rows, ALD was stained with AlexaFluor 488 and PEX11 was detected by a Cy3-coupled secondary antibody. In the bottom row, PEX11 was Alexa-stained and HXK detected by Cy3. Corresponding Nomarski images and DAPI staining are shown on the left. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 1. (A) Northern blot. Double-stranded RNA causes destruction of PEX2 mRNA. Each lane on the blot contains 10 µg poly(A)+ RNA from the procyclic form RNAi cell line. Cells were grown either without Tet (-), or for 13 hours in the presence of 100 ng/ml Tet (+). (B) Growth curves. Filled figures and unbroken lines, cells grown without Tet; open figures and dashed lines, cells grown in the presence of 100 ng/ml Tet. Diamonds, cells with pex2r downstream of the inducible RNA polymerase I promoter. The cultures were initiated at 1.5x105 cells/ml and followed for 2 days. Squares, cells expressing the T7 polymerase and with a full-length PEX2 downstream of an inducible T7 promoter. Circles, the dominant-negative mutant — cells expressing the T7 polymerase and with the pex2r gene downstream of an inducible T7 promoter. Cultures of cells expressing the T7 polymerase were initiated at 5x104 cells/ml. (C) Western blot. Digitonin treatment of the dominant-negative mutant. 10 µg of protein (1-2x106 cells) were taken from each sample (cells grown with and without Tet) for treatment with 6 µg of digitonin. s, supernatant (cytosolic fraction); p, pellet (glycosomal fraction). The glycosomal proteins detected are indicated on the right. (D) Growth curves of RNA interference cells. Filled circles and unbroken lines, control — cells grown without Tet; open circles and broken lines, cells in the presence of 100 ng/ml Tet. Bloodstream cultures were initiated at 1x105 cells/ml and daily diluted to the same concentration. Procyclic cultures were daily diluted to 5x105 cells/ml.

View larger version (58K): [in a new window]   Fig. 3. Effects of PEX2 depletion on matrix protein import and glycosome morphology in procyclic trypanosomes. DAPI staining is shown in blue, PEX11 in green and GAPDH in red. Cells were grown in the presence (+Tet) or absence (-Tet) of 100 ng/ml Tet for 3 days. Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 4. (A) Effects of PEX2 depletion on the location of HXK and PGI activities. Bloodstream and procyclic pex2 cells were disrupted using glass beads. Enzymatic activities were measured in the cytosolic (black) and organellar pellet (gray) fractions. The +Tet columns are from cells grown in the presence of 100 ng/ml Tet for 36 hours; -Tet columns are the controls. Measurements were made with three independent cell extracts; the activities shown are the mean and standard deviation from two to five readings. (B) Western blots showing the distribution of proteins in procyclic mutants, 0, 1, 2 and 3 days after Tet addition. Cells were separated into digitonin-soluble (s) and pellet (p) fractions. The proteins detected are indicated on the right. (C) Western blot showing the expression of EP1 in procyclic mutants grown in the absence and in the presence of Tet for up to 3 days. Each lane was loaded with a total protein extract from 5x105 cells.

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