Regulation of the translocation efficiency of Icm/Dot translocated substrates by protein conformation.
Amyot, Whitney.
2012
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Abstract: Legionella
pneumophila uses a type 4B secretion system known as the Icm/Dot system to deliver over
200 effector proteins to the host cell for the establishment and maintenance of a
replication vacuole. Translocation of a large majority of these Icm/Dot translocated
substrates (IDTS) is facilitated by the Icm/Dot adapter complex IcmS/W. The
characteristics of translocation competence... read morefor substrates of the Icm/Dot secretion
system are poorly elucidated. In these studies, I investigated the effect of protein
conformation on Icm/Dot translocation efficiency by analyzing the translocation of
stably folded IDTS fusions and the interaction of IDTS with the chaperone protein
complex IcmS/W. IDTS fused to dihydrofolate reductase (DHFR) or to folded ubiquitin (Ub)
exhibited a kinetic defect in translocation, indicating that substrates of the Icm/Dot
T4SS are translocated to the host cytosol in an extended or unfolded conformation.
Additionally, expression of folded substrates decreased the translocation of at least
one IDTS, suggesting that these proteins are blocking the translocon or impairing the
function of the Icm/Dot complex. The requirement of IcmS/W for translocation of IDTS was
also characterized as a kinetic response that varied between IDTS, suggesting a role of
IcmS/W in the temporal regulation of translocation. Deletion analysis of the IDTS MavU
and SidG failed to identify a specific site for IcmS/W binding. Rather, IcmS/W
interacted with multiple regions within the IDTS as well as the enzymatic domain of
CyaA, the adenylate cyclase toxin of Bordetella pertussis. IcmS/W also facilitates the
transport or recognition of folded proteins, as DHFR fusions exhibit a severe
translocation defect in an icmS- mutant strain. These data are consistent with a role
for IcmS in promoting translocation efficiency by non-specifically binding to proteins
to hold them in an elongated or partially unfolded conformation for recognition by the
Icm/Dot complex.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Ralph Isberg.
Committee: Carol Kumamoto, Abraham Sonenshein, Joan Mecsas, and Joseph Vogel.
Keyword: Microbiology.read less - ID:
- q524k1238
- Component ID:
- tufts:20231
- To Cite:
- DCA Citation Guide EndNote
