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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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fla15 was defective for two polypeptides of the 17S sedimenting fractions from the cytoplasmic matrix of flagella. Autoradiograms of 35S-labeled polypeptides contained in sucrose gradient fractions 6–16 after separation by gel electrophoresis.  Molecular weight standards are indicated on the left. (a) Proteins  from a wild-type strain. Lines between lanes 9 and 10 indicate the  presence of components 1–13 of the two 17S complexes. (b) Proteins from fla15. Asterisks between lanes 9 and 10 indicate the  position of the two polypeptides that are deficient in fla15. Lines  indicate the rest of the subunits of 17S complexes.
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Figure 5: fla15 was defective for two polypeptides of the 17S sedimenting fractions from the cytoplasmic matrix of flagella. Autoradiograms of 35S-labeled polypeptides contained in sucrose gradient fractions 6–16 after separation by gel electrophoresis. Molecular weight standards are indicated on the left. (a) Proteins from a wild-type strain. Lines between lanes 9 and 10 indicate the presence of components 1–13 of the two 17S complexes. (b) Proteins from fla15. Asterisks between lanes 9 and 10 indicate the position of the two polypeptides that are deficient in fla15. Lines indicate the rest of the subunits of 17S complexes.

Mentions: The sedimentation profiles of 35S-labeled flagellar proteins from mutant strains were indistinguishable from that of a wild-type strain. However, the electrophoretograms of protein fractions from the sucrose gradients revealed that the 17S complexes from each mutant were deficient to a different extent for the same two polypeptides with apparent molecular weights of 148,000 and 127,000. The electrophoretic bands of defective polypeptides from fla15 are indicated by asterisks located between lanes 9 and 10 of the electrophoretograms (Fig. 5 b). The other subunits of 17S complexes from fla15 and a wild-type strain are indicated by lines (Fig. 5, b and a, respectively).


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)

fla15 was defective for two polypeptides of the 17S sedimenting fractions from the cytoplasmic matrix of flagella. Autoradiograms of 35S-labeled polypeptides contained in sucrose gradient fractions 6–16 after separation by gel electrophoresis.  Molecular weight standards are indicated on the left. (a) Proteins  from a wild-type strain. Lines between lanes 9 and 10 indicate the  presence of components 1–13 of the two 17S complexes. (b) Proteins from fla15. Asterisks between lanes 9 and 10 indicate the  position of the two polypeptides that are deficient in fla15. Lines  indicate the rest of the subunits of 17S complexes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132975&req=5

Figure 5: fla15 was defective for two polypeptides of the 17S sedimenting fractions from the cytoplasmic matrix of flagella. Autoradiograms of 35S-labeled polypeptides contained in sucrose gradient fractions 6–16 after separation by gel electrophoresis. Molecular weight standards are indicated on the left. (a) Proteins from a wild-type strain. Lines between lanes 9 and 10 indicate the presence of components 1–13 of the two 17S complexes. (b) Proteins from fla15. Asterisks between lanes 9 and 10 indicate the position of the two polypeptides that are deficient in fla15. Lines indicate the rest of the subunits of 17S complexes.
Mentions: The sedimentation profiles of 35S-labeled flagellar proteins from mutant strains were indistinguishable from that of a wild-type strain. However, the electrophoretograms of protein fractions from the sucrose gradients revealed that the 17S complexes from each mutant were deficient to a different extent for the same two polypeptides with apparent molecular weights of 148,000 and 127,000. The electrophoretic bands of defective polypeptides from fla15 are indicated by asterisks located between lanes 9 and 10 of the electrophoretograms (Fig. 5 b). The other subunits of 17S complexes from fla15 and a wild-type strain are indicated by lines (Fig. 5, b and a, respectively).

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