Identifying a Targeting Factor for Rab27a
Author(s)
Booth, Antonia Elspeth Grace
Type
Thesis
Abstract
The Rab family of small GTPases regulate intracellular membrane trafficking pathways and each Rab GTPase localises to a discrete membrane compartment. The mechanism(s) by which this targeting is achieved remains to be elucidated. Previous work identified that the C‐terminal hypervariable domain and effector binding were not necessary for Rab27a targeting in melanocytes. However, GEF activity of the non‐redundant Rab27a GEF Rab3GEP (R3G) was required but not sufficient for Rab27a targeting. This led to the hypothesis of the involvement of a Rab27a targeting factor.
A Rab27a mutant (Rab27aSF1/F4) had previously been characterised that maintained melanosomal targeting and GTP‐loading by R3G but bound to no known Rab27a effectors. Using this mutant, novel Rab27a interacting partners were identified and assessed for their possible role in Rab27a targeting. The current research identified ATP1a1 (the α1 subunit of Na+,K+‐ATPase) as a novel interacting partner for Rab27a in melanocytes, depletion of which resulted in perinuclear clustering of melanosomes, indicative of loss of Rab27a function. Depletion of ATP1a1 did not disrupt Rab27a membrane localisation but did reduce the levels of GTP‐bound Rab27a, comparable to what is seen following depletion of R3G. Crucially, depletion of ATP1a1 resulted in displacement of endogenous Rab27a from melanosomal membranes indicating a role in Rab27a targeting. Furthermore, ATP1a1 depletion disrupted the levels of other melanosomal proteins indicative of a disruption in melanogenesis. ATP1a1 is proposed to transiently associate with melanosomes and through its Na+,K+‐ATPase pump function regulate the lumenal pH, which indirectly regulates melanosome maturation. How the maturation of the lumenal environment is conveyed to Rab27a, and is subsequent recruitment to melanosomes remains to be clarified.
A Rab27a mutant (Rab27aSF1/F4) had previously been characterised that maintained melanosomal targeting and GTP‐loading by R3G but bound to no known Rab27a effectors. Using this mutant, novel Rab27a interacting partners were identified and assessed for their possible role in Rab27a targeting. The current research identified ATP1a1 (the α1 subunit of Na+,K+‐ATPase) as a novel interacting partner for Rab27a in melanocytes, depletion of which resulted in perinuclear clustering of melanosomes, indicative of loss of Rab27a function. Depletion of ATP1a1 did not disrupt Rab27a membrane localisation but did reduce the levels of GTP‐bound Rab27a, comparable to what is seen following depletion of R3G. Crucially, depletion of ATP1a1 resulted in displacement of endogenous Rab27a from melanosomal membranes indicating a role in Rab27a targeting. Furthermore, ATP1a1 depletion disrupted the levels of other melanosomal proteins indicative of a disruption in melanogenesis. ATP1a1 is proposed to transiently associate with melanosomes and through its Na+,K+‐ATPase pump function regulate the lumenal pH, which indirectly regulates melanosome maturation. How the maturation of the lumenal environment is conveyed to Rab27a, and is subsequent recruitment to melanosomes remains to be clarified.
Date Issued
2012
Date Awarded
2012-07
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Seabra, Miguel
Leitinger, Birgit
Publisher Department
National Heart and Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)