Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval
Matthew N. J. Seaman
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University of Cambridge, Cambridge Institute for Medical Research/Clinical Biochemistry, Wellcome Trust/MRC building, Addenbrookes Hospital
,
Cambridge, CB2 0XY
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UK
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Summary
The cation-independent mannose 6-phosphate receptor
(CIMPR) cycles between the trans-Golgi network (TGN)
and endosomes to mediate sorting of lysosomal hydrolases.
The endosome-to-TGN retrieval of the CIMPR requires the
retromer complex. Genetic, biochemical and structural
data support the hypothesis that the retromer can directly
bind to the tail of the CIMPR, to sort the CIMPR into
ce vesicles and tubules for retrieval to the TGN. Presently,
n however, no known retromer sorting motif in the tail of the
e
ic CIMPR has been identified. Using CD8-reporter proteins
S carrying the cytoplasmic tail of the CIMPR we have
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l Introduction
a
n The function of the different organelles that comprise the
ru secretory and endocytic pathways in eukaryotic cells is
o determined by the complement of resident proteins present
J
within the respective organelle. The mechanisms that govern
membrane protein localisation to the different organelles rely
on intrinsic information in the membrane protein such as
sorting motifs in the cytoplasmic domain and extrinsic
factors such as coat-proteins that recognise the sorting
motifs.
A well-studied example of these concepts is the sorting of
membrane proteins by the clathrin-coated vesicle (CCV)
adaptor proteins through recognition of the YXX sorting
motif. This tetra-peptide motif, comprising Tyr-X-X (where
X is any amino acid)-bulky hydrophobic, is bound by the
medium chain ( ) subunit of adaptor complexes resulting in
the protein containing the YXX motif being concentrated
in CCVs (Ohno et al., 1995; Owen and Evans, 1998)
(reviewed in Bonifacino and Traub, 2003). Another example
of sorting motifs interacting with coat proteins is the
recognition of acidic di-leucine motifs by Golgi-associated
ear-containing ARF-binding (GGA) proteins. GGA proteins
bind to the consensus sequence, Asp-X-X-Leu-Leu (DXXLL)
and function at the trans-Golgi network (TGN) to direct
specific cargo proteins into CCVs (Puertollano et al., 2001;
Misra et al., 2002). Sorting motifs, therefore, are often just
two to four amino acids present in the correct molecular
context. YXX motifs must be at least seven amino acids
from the transmembrane domain (Rohrer et al., 1996)
whereas acidic di-leucine motifs are usually near the end of
systematically dissected the CIMPR tail to identify a novel,
conserved aromatic-containing sorting motif that is critical
for the endosome-to-TGN retrieval of the CIMPR and for
the interaction with retromer and the clathrin adaptor
AP-1.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/14/2378/DC1
the cytoplasmic domain of proteins that traffic between the
TGN and endosomes.
Membrane proteins that traffic in the post-Golgi
endomembrane system often contain multiple sorting motifs that
function to direct the transport of the membrane protein
between the various compartments such as the TGN,
endosomes and the plasma membrane. A good example of
this is the cation-independent mannose 6-phosphate receptor
(CIMPR) which binds to lysosomal hydrolases in the TGN
and is sorted into CCVs by GGA proteins in concert with the
AP-1 clathrin adaptor (Ghosh et al., 2003; Doray et al., 2002).
The cytoplasmic tail of the CIMPR, therefore, has both
YXX and DXXLL motifs. In the case of the CIMPR, the
affinity of the GGAs for DXXLL motifs is augmented by
casein kinase-mediated phosphorylation of a serine residue
that immediately precedes the aspartate of the acidic
dileucine (Kato et al., 2002). Following the delivery of the
CIMPR to an endosome, the CIMPR releases its ligand and
is then recycled back to the TGN to undergo further rounds
of hydrolase sorting. Retrieval of the CIMPR from the
endosome to the TGN is mediated by the retromer complex
(Seaman, 2004; Arighi et al., 2004; Carlton et al., 2004) and
has also been shown to require the function of the TIP47 and
PACS1 proteins (reviewed by Seaman, 2005; Bonifacino and
Rojas, 2006).
Retromer was first described in yeast and is required for
the endosome-to-Golgi retrieval of the vacuolar hydrolase
receptor Vps10p, the yeast functional equivalent of the
CIMPR (Seaman et al., 1997; Seaman et al., 1998). Retromer
comprises five proteins in yeast, Vps35p, Vps29p, Vps26p,
Vps5p and Vps17p, which are conserved in mammals (with
the exception of Vps17p that has no clear homologue)
(reviewed by Seaman, 2005). Retromer can recognise cargo
such as Vps10p and the CIMPR through interactions with
Vps35p or mammalian VPS35, respectively (Nothwehr et
al., 2000; Arighi et al., 2004). Retromer can also reshape the
membrane by the action of the sorting nexin components
Vps5p and its mammalian homologue, sorting nexin-1
(SNX1) (Seaman et al., 1998; Carlton et al., 2004). Recent
structural and biochemical studies of VPS29 h (...truncated)