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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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17S sedimenting fractions from fla15 and fla17-1 were deficient for the same four polypeptides to a different extent. Autoradiographs of two-dimensional maps of 35S-labeled polypeptides contained in 17S sedimenting fractions from flagella of (a) wild-type, (b)  fla15, and (c) fla17-1. Numbers and lines at the left side of a refer to the position of subunits of the 17S complexes, as determined by one-dimensional electrophoresis. Oblique lines in each panel indicate the four polypeptides that are deficient in fla15 and fla17-1. The new  polypeptide present in the 17S sedimenting fractions from fla17-1 is indicated by an arrowhead in c. Polypeptides appear in the maps in  increasing order of acidity from left to right.
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Figure 7: 17S sedimenting fractions from fla15 and fla17-1 were deficient for the same four polypeptides to a different extent. Autoradiographs of two-dimensional maps of 35S-labeled polypeptides contained in 17S sedimenting fractions from flagella of (a) wild-type, (b) fla15, and (c) fla17-1. Numbers and lines at the left side of a refer to the position of subunits of the 17S complexes, as determined by one-dimensional electrophoresis. Oblique lines in each panel indicate the four polypeptides that are deficient in fla15 and fla17-1. The new polypeptide present in the 17S sedimenting fractions from fla17-1 is indicated by an arrowhead in c. Polypeptides appear in the maps in increasing order of acidity from left to right.

Mentions: Comparison of two-dimensional electrophoretograms of polypeptides from fla15 and a wild-type strain revealed that the mutant lacked the majority of two polypeptides of apparent molecular weight 148,000, as well as polypeptides of molecular weights 127,000 and 43,000, respectively (Table III). These four polypeptides are indicated by oblique lines in the maps of polypeptides from wild-type and fla15 (Fig. 7, a and b). Two-dimensional maps of polypeptides from fla17-1 had reduced amounts of the same four polypeptides defective in fla15 (Fig. 7, c and b). The new 135,000 apparent molecular weight polypeptide present in fla17-1 is indicated by an arrowhead in Fig. 7 c. The two-dimensional map of polypeptides from fla16 was indistinguishable from that of fla17-1.


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)

17S sedimenting fractions from fla15 and fla17-1 were deficient for the same four polypeptides to a different extent. Autoradiographs of two-dimensional maps of 35S-labeled polypeptides contained in 17S sedimenting fractions from flagella of (a) wild-type, (b)  fla15, and (c) fla17-1. Numbers and lines at the left side of a refer to the position of subunits of the 17S complexes, as determined by one-dimensional electrophoresis. Oblique lines in each panel indicate the four polypeptides that are deficient in fla15 and fla17-1. The new  polypeptide present in the 17S sedimenting fractions from fla17-1 is indicated by an arrowhead in c. Polypeptides appear in the maps in  increasing order of acidity from left to right.
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Related In: Results  -  Collection

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Figure 7: 17S sedimenting fractions from fla15 and fla17-1 were deficient for the same four polypeptides to a different extent. Autoradiographs of two-dimensional maps of 35S-labeled polypeptides contained in 17S sedimenting fractions from flagella of (a) wild-type, (b) fla15, and (c) fla17-1. Numbers and lines at the left side of a refer to the position of subunits of the 17S complexes, as determined by one-dimensional electrophoresis. Oblique lines in each panel indicate the four polypeptides that are deficient in fla15 and fla17-1. The new polypeptide present in the 17S sedimenting fractions from fla17-1 is indicated by an arrowhead in c. Polypeptides appear in the maps in increasing order of acidity from left to right.
Mentions: Comparison of two-dimensional electrophoretograms of polypeptides from fla15 and a wild-type strain revealed that the mutant lacked the majority of two polypeptides of apparent molecular weight 148,000, as well as polypeptides of molecular weights 127,000 and 43,000, respectively (Table III). These four polypeptides are indicated by oblique lines in the maps of polypeptides from wild-type and fla15 (Fig. 7, a and b). Two-dimensional maps of polypeptides from fla17-1 had reduced amounts of the same four polypeptides defective in fla15 (Fig. 7, c and b). The new 135,000 apparent molecular weight polypeptide present in fla17-1 is indicated by an arrowhead in Fig. 7 c. The two-dimensional map of polypeptides from fla16 was indistinguishable from that of fla17-1.

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