Rab27a Targeting to Melanosomes Requires Nucleotide Exchange but not Effector Binding.

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Abstract

Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently each Rab occupies a highly specific subcellular localisation. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterised extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC, et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J. Biol. Chem. 2008;283(34):23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mistargeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.
Original languageEnglish
Pages (from-to)1056-1066
Number of pages11
JournalTraffic -Copenhagen then Oxford-
Volume12
Issue numberNA
DOIs
Publication statusPublished - 2011

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Melanosomes
Guanine Nucleotide Exchange Factors
Nucleotides
Melanocytes
Organelles
rab GTP-Binding Proteins
Membranes
Secretory Vesicles
Protein Transport
Guanosine Triphosphate
Lysosomes

Cite this

@article{99ae39428e434e08bf6da02a237b1413,
title = "Rab27a Targeting to Melanosomes Requires Nucleotide Exchange but not Effector Binding.",
abstract = "Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently each Rab occupies a highly specific subcellular localisation. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterised extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC, et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J. Biol. Chem. 2008;283(34):23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mistargeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.",
keywords = "DENN DOMAIN, TRANSPORT, EARLY ENDOSOMES, GRISCELLI-SYNDROME, MELANOCYTES, effectors, guanine nucleotide exchange factor, RECRUITMENT, targeting, SPECIFICITY, MYOSIN VA, FAMILY, Rab, GTPASES, melanosome",
author = "Ramalho, {Jos{\'e} da Silva} and Miguel Seabra",
year = "2011",
doi = "10.1111/j.1600-0854.2011.01216.x",
language = "English",
volume = "12",
pages = "1056--1066",
journal = "Traffic -Copenhagen then Oxford-",
issn = "1398-9219",
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number = "NA",

}

TY - JOUR

T1 - Rab27a Targeting to Melanosomes Requires Nucleotide Exchange but not Effector Binding.

AU - Ramalho, José da Silva

AU - Seabra, Miguel

PY - 2011

Y1 - 2011

N2 - Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently each Rab occupies a highly specific subcellular localisation. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterised extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC, et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J. Biol. Chem. 2008;283(34):23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mistargeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.

AB - Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently each Rab occupies a highly specific subcellular localisation. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterised extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC, et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J. Biol. Chem. 2008;283(34):23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mistargeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.

KW - DENN DOMAIN

KW - TRANSPORT

KW - EARLY ENDOSOMES

KW - GRISCELLI-SYNDROME

KW - MELANOCYTES

KW - effectors

KW - guanine nucleotide exchange factor

KW - RECRUITMENT

KW - targeting

KW - SPECIFICITY

KW - MYOSIN VA

KW - FAMILY

KW - Rab

KW - GTPASES

KW - melanosome

U2 - 10.1111/j.1600-0854.2011.01216.x

DO - 10.1111/j.1600-0854.2011.01216.x

M3 - Article

VL - 12

SP - 1056

EP - 1066

JO - Traffic -Copenhagen then Oxford-

JF - Traffic -Copenhagen then Oxford-

SN - 1398-9219

IS - NA

ER -