The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers.

REFERENCES

al-Shammary
F. J.
,
Shoukrey
N. M.
,
al-Shewemi
S. E.
,
Ibrahim
E. A.
,
al-Zahrani
M. A.
,
al-Tuwaijri
A. S.
(
1995
).
Leishmania major: in vitro ultrastructural study of the paraxial rod of promastigotes.
Int. J. Parasitol
25
,
443
452
Bastin
P.
,
Bagherzadeh
K.
,
Matthews
K. R.
,
Gull
K.
(
1996
).
A novel epitope tag system to study protein targeting and organelle biosynthesis in Trypanosoma cruzi.
Mol. Biochem. Parasitol
77
,
235
239
Bastin
P.
,
Sherwin
T.
,
Gull
K.
(
1998
).
Paraflagellar rod is vital for Trypanosome motility [letter].
Nature
391
,
548
–.
Beard
C. A.
,
Saborio
J. L.
,
Tewari
D.
,
Krieglstein
K. G.
,
Henschen
A. H.
,
Manning
J. E.
(
1992
).
Evidence for two distinct major protein components, PAR 1 and PAR 2, in the paraflagellar rod of Trypanosoma cruzi. Complete nucleotide sequence of PAR 2.
J. Biol. Chem
267
,
21656
21662
Beverley
S. M.
(
1988
).
Characterization of the unusual mobility of large circular DNAs in pulsed field-gradient electrophoresis.
Nucl. Acids Res
16
,
925
938
Burland
T. G.
,
Gull
K.
,
Schedl
T.
,
Boston
R. S.
,
Dove
W. F.
(
1983
).
Cell type-dependent expression of tubulins in Physarum.
J. Cell Biol
97
,
1852
1859
Cruz
A.
,
Coburn
C. M.
,
Beverley
S. M.
(
1991
).
Double targeted gene replacement for creating null mutants.
Proc. Nat. Acad. Sci. USA
88
,
7170
7174
Deflorin
J.
,
Rudolf
M.
,
Seebeck
T.
(
1994
).
The major components of the paraflagellar rod of Trypanosoma brucei are two similar, but distinct proteins which are encoded by two different gene loci.
J. Biol. Chem
269
,
28745
28751
Dutcher
S. K.
(
1995
).
Flagellar assembly in two hundred and fifty easy-to-follow steps.
Trends Genet
11
,
398
404
Farina
M.
,
Attias
M.
,
Suoto-Padron
T.
,
De Souza
W.
(
1986
).
Further studies on the organization of the paraxial rod of Trypanosomatids.
J. Protozool
33
,
552
557
Fouts
D. L.
,
Stryker
G. A.
,
Gorski
K. S.
,
Miller
M. J.
,
Nguyen
T. V.
,
Wrightsman
R. A.
,
Manning
J. E.
(
1998
).
Evidence for four distinct major protein components in the paraflagellar rod of Trypanosoma cruzi.
J. Biol. Chem
273
,
21846
21855
Freedman
D. J.
,
Beverley
S. M.
(
1993
).
Two more independent selectable markers for stable transfection of Leishmania.
Mol. Biochem. Parasitol
62
,
37
44
Gueiros-Filho
F. J.
,
Beverley
S. M.
(
1996
).
Selection against the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major.
Mol. Cell. Biol
16
,
5655
5663
Hyams
J. S.
(
1982
).
The Euglena paraflagellar rod: structure, relationship to other flagellar components and preliminary biochemical characterization.
J. Cell Sci
55
,
199
210
Imboden
M.
,
Muller
N.
,
Hemphill
A.
,
Mattioli
R.
,
Seebeck
T.
(
1995
).
Repetitive proteins from the flagellar cytoskeleton of African trypanosomes are diagnostically useful antigens.
Parasitology
110
,
249
258
Ismach
R.
,
Cianci
C. M.
,
Caulfield
J. P.
,
Langer
P. J.
,
Hein
A.
,
McMahon-Pratt
D.
(
1989
).
Flagellar membrane and paraxial rod proteins of Leishmania: characterization employing monoclonal antibodies.
J. Protozool
36
,
617
624
Johnson
K. A.
,
Rosenbaum
J. L.
(
1992
).
Polarity of flagellar assembly in Chlamydomonas.
J. Cell Biol
119
,
1605
1611
Joshi
P. B.
,
Webb
J. R.
,
Davies
J. E.
,
McMaster
W. R.
(
1995
).
The gene encoding streptothricin acetyltransferase (sat) as a selectable marker for Leishmania expression vectors.
Gene
156
,
145
149
Kelly
J. M.
,
Ward
H. M.
,
Miles
M. A.
,
Kendall
G.
(
1992
).
A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania.
Nucl. Acids Res
20
,
3963
3969
Kohl
L.
,
Sherwin
T.
,
Gull
K.
(
1999
).
Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle.
J. Euk. Microbiol
46
,
105
109
LeBowitz
J. H.
,
Coburn
C. M.
,
Beverley
S. M.
(
1991
).
Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes.
Gene
103
,
119
123
Moore
L. L.
,
Santrich
C.
,
LeBowitz
J. H.
(
1996
).
Stage-specific expression of the Leishmania mexicana paraflagellar rod protein PFR-2.
Mol. Biochem. Parasitol
80
,
125
135
Ngo
H. M.
,
Bouck
G. B.
(
1998
).
Heterogeneity and a coiled coil prediction of trypanosomatid-like flagellar rod proteins in Euglena.
J. Eukaryot. Microbiol
45
,
323
333
Rosenbaum
J. L.
,
Cole
D. G.
,
Diener
D. R.
(
1999
).
Intraflagellar transport: the eyes have it.
J. Cell Biol
144
,
385
388
Russell
D. G.
,
Newsam
R. J.
,
Palmer
G. C.
,
Gull
K.
(
1983
).
Structural and biochemical characterisation of the paraflagellar rod of Crithidia fasciculata.
Eur. J. Cell Biol
30
,
137
143
Saborio
J. L.
,
Manuel Hernandez
J.
,
Narayanswami
S.
,
Wrightsman
R.
,
Palmer
E.
,
Manning
J.
(
1989
).
Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi.
J. Biol. Chem
264
,
4071
4075
Santrich
C.
,
Moore
L.
,
Sherwin
T.
,
Bastin
P.
,
Brokaw
C.
,
Gull
K.
,
LeBowitz
J. H.
(
1997
).
A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.
Mol. Biochem. Parasitol
90
,
95
109
van Tuinen
E.
,
Riezman
H.
(
1987
).
Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.
J. Histochem. Cytochem
35
,
327
333
Woods
A.
,
Sherwin
T.
,
Sasse
R.
,
MacRae
T. H.
,
Baines
A. J.
,
Gull
K.
(
1989
).
Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.
J. Cell Sci
93
,
491
500
Woodward
R.
,
Carden
M. J.
,
Gull
K.
(
1994
).
Molecular characterisation of a novel, repetitive protein of the paraflagellar rod in Trypanosoma brucei.
Mol. Biochem. Parasitol
67
,
31
39
This content is only available via PDF.