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Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects.

Piperno G, Siuda E, Henderson S, Segil M, Vaananen H, Sassaroli M - J. Cell Biol. (1998)

Bottom Line: Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images.Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A.The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, 10029, USA. Piperno@msvax.mssm.edu

ABSTRACT
A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

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The four polypeptides that are deficient from flagella of  fla15, fla16, and fla17-1 behaved as subunits of a complex. Autoradiography of a two-dimensional map of 35S-labeled polypeptides  from flagella of a wild-type strain after subsequent sedimentation  in sucrose gradient and chromatography in a DEAE-Sepharose  column. Oblique lines indicate the four polypeptides that are deficient in fla15, fla16, and fla17-1. Polypeptides appear in the map  in increasing order of acidity from left to right.
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Figure 8: The four polypeptides that are deficient from flagella of fla15, fla16, and fla17-1 behaved as subunits of a complex. Autoradiography of a two-dimensional map of 35S-labeled polypeptides from flagella of a wild-type strain after subsequent sedimentation in sucrose gradient and chromatography in a DEAE-Sepharose column. Oblique lines indicate the four polypeptides that are deficient in fla15, fla16, and fla17-1. Polypeptides appear in the map in increasing order of acidity from left to right.

Mentions: To determine whether these four polypeptides remain associated in a complex after chromatography and exposure to a high concentration of salt, we performed one- and two-dimensional electrophoresis of wild-type proteins that were eluted by a gradient of NaCl at pH 6.8 from a DEAE-Sepharose column. At least three protein fractions eluted at 0.29–0.30 M NaCl had identical compositions, as assessed by one-dimensional electrophoresis. Polypeptides eluted in that range of NaCl molarity formed a map (Fig. 8) that was simpler than that shown in Fig. 7 a but still included the four polypeptides that were deficient from flagella of fla15, fla16, and fla17-1 (Fig. 8), indicated by oblique lines. Therefore, these polypeptides behaved as subunits of a complex that was stable after two subsequent procedures of protein purification.


Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects.

Piperno G, Siuda E, Henderson S, Segil M, Vaananen H, Sassaroli M - J. Cell Biol. (1998)

The four polypeptides that are deficient from flagella of  fla15, fla16, and fla17-1 behaved as subunits of a complex. Autoradiography of a two-dimensional map of 35S-labeled polypeptides  from flagella of a wild-type strain after subsequent sedimentation  in sucrose gradient and chromatography in a DEAE-Sepharose  column. Oblique lines indicate the four polypeptides that are deficient in fla15, fla16, and fla17-1. Polypeptides appear in the map  in increasing order of acidity from left to right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132975&req=5

Figure 8: The four polypeptides that are deficient from flagella of fla15, fla16, and fla17-1 behaved as subunits of a complex. Autoradiography of a two-dimensional map of 35S-labeled polypeptides from flagella of a wild-type strain after subsequent sedimentation in sucrose gradient and chromatography in a DEAE-Sepharose column. Oblique lines indicate the four polypeptides that are deficient in fla15, fla16, and fla17-1. Polypeptides appear in the map in increasing order of acidity from left to right.
Mentions: To determine whether these four polypeptides remain associated in a complex after chromatography and exposure to a high concentration of salt, we performed one- and two-dimensional electrophoresis of wild-type proteins that were eluted by a gradient of NaCl at pH 6.8 from a DEAE-Sepharose column. At least three protein fractions eluted at 0.29–0.30 M NaCl had identical compositions, as assessed by one-dimensional electrophoresis. Polypeptides eluted in that range of NaCl molarity formed a map (Fig. 8) that was simpler than that shown in Fig. 7 a but still included the four polypeptides that were deficient from flagella of fla15, fla16, and fla17-1 (Fig. 8), indicated by oblique lines. Therefore, these polypeptides behaved as subunits of a complex that was stable after two subsequent procedures of protein purification.

Bottom Line: Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images.Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A.The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, 10029, USA. Piperno@msvax.mssm.edu

ABSTRACT
A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

Show MeSH
Related in: MedlinePlus