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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, fla16 and fla17-1 were defective to different extents in the same three polypeptides. Autoradiograms of 35S-labeled polypeptides of 17S sedimenting fractions from flagella  of wild-type and mutant strains following one-dimensional electrophoresis at high resolution. First lane in a and b, proteins from  a wild-type strain. Second lane in a, proteins from fla15. Second  and third lane in b, proteins from fla16 and fla17-1. Equal  amounts of cpm were analyzed in each lane. Numbers and lines at  the left of the figure refer to 13 subunits of 17S complexes. Lines  between the two panels indicate the electrophoretic bands that  are deficient in each mutant and represent putative subunits of  complex A. A dot between the second and third lane in b refers  to a polypeptide that is present only in 17S sedimenting fractions  from fla16 and fla17-1. Molecular weights of standards are indicated on the right side.
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Figure 6: fla15, fla16 and fla17-1 were defective to different extents in the same three polypeptides. Autoradiograms of 35S-labeled polypeptides of 17S sedimenting fractions from flagella of wild-type and mutant strains following one-dimensional electrophoresis at high resolution. First lane in a and b, proteins from a wild-type strain. Second lane in a, proteins from fla15. Second and third lane in b, proteins from fla16 and fla17-1. Equal amounts of cpm were analyzed in each lane. Numbers and lines at the left of the figure refer to 13 subunits of 17S complexes. Lines between the two panels indicate the electrophoretic bands that are deficient in each mutant and represent putative subunits of complex A. A dot between the second and third lane in b refers to a polypeptide that is present only in 17S sedimenting fractions from fla16 and fla17-1. Molecular weights of standards are indicated on the right side.

Mentions: To test these hypotheses and determine whether other deficiencies occur in each mutant, we separated the polypeptides of 17S sedimenting fractions from wild-type and mutant strains by high-resolution one- and two dimensional gel electrophoresis. In these experiments, we identified the 13 subunits of the two 17S complexes by their apparent molecular weights. These subunits are indicated by lines and progressive numbers in Fig. 6.


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, fla16 and fla17-1 were defective to different extents in the same three polypeptides. Autoradiograms of 35S-labeled polypeptides of 17S sedimenting fractions from flagella  of wild-type and mutant strains following one-dimensional electrophoresis at high resolution. First lane in a and b, proteins from  a wild-type strain. Second lane in a, proteins from fla15. Second  and third lane in b, proteins from fla16 and fla17-1. Equal  amounts of cpm were analyzed in each lane. Numbers and lines at  the left of the figure refer to 13 subunits of 17S complexes. Lines  between the two panels indicate the electrophoretic bands that  are deficient in each mutant and represent putative subunits of  complex A. A dot between the second and third lane in b refers  to a polypeptide that is present only in 17S sedimenting fractions  from fla16 and fla17-1. Molecular weights of standards are indicated on the right side.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132975&req=5

Figure 6: fla15, fla16 and fla17-1 were defective to different extents in the same three polypeptides. Autoradiograms of 35S-labeled polypeptides of 17S sedimenting fractions from flagella of wild-type and mutant strains following one-dimensional electrophoresis at high resolution. First lane in a and b, proteins from a wild-type strain. Second lane in a, proteins from fla15. Second and third lane in b, proteins from fla16 and fla17-1. Equal amounts of cpm were analyzed in each lane. Numbers and lines at the left of the figure refer to 13 subunits of 17S complexes. Lines between the two panels indicate the electrophoretic bands that are deficient in each mutant and represent putative subunits of complex A. A dot between the second and third lane in b refers to a polypeptide that is present only in 17S sedimenting fractions from fla16 and fla17-1. Molecular weights of standards are indicated on the right side.
Mentions: To test these hypotheses and determine whether other deficiencies occur in each mutant, we separated the polypeptides of 17S sedimenting fractions from wild-type and mutant strains by high-resolution one- and two dimensional gel electrophoresis. In these experiments, we identified the 13 subunits of the two 17S complexes by their apparent molecular weights. These subunits are indicated by lines and progressive numbers in Fig. 6.

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