www.elsevier.com/locate/yexcr

Experimental Cell Resear

Characterization of the human GARP

(Golgi associated retrograde protein) complex$

Heike Liewena, Ivo Meinhold-Heerleinb, Vasco Oliveirac, Robert Schwarzenbacherc,

Guorong Luod, Andreas Wadlea, Martin Junge, Michael Pfreundschuha, Frank Stenner-Liewena,TaMedical Department I, University of the Saarland, Homburg 66421, Germany

bDepartment of Obstetrics and Gynecology, University of Kiel, 24105 Kiel, GermanycThe Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USAdDepartment of Histology and Embryology, Nanning, Guangxi 530021, PR China

eDepartment of Biochemistry, University of the Saarland, Homburg 66421, Germany

Received 21 June 2004, revised version received 24 January 2005

Available online 9 March 2005

Abstract

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental

transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells.

Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol.

Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining

experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive

endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated

transport processes.

In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate

membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52.

In human cells, the borphanQ SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously

unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant

conservation between various species but diversification and specialization result in important differences in human cells.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Human Golgi associated retrograde protein complex; GARP; Human Vps fifty-three complex (VFT); Tethering factor; Human Vps54; Rab6;

Syntaxin 10; SNARE; Coiled-coil domains; Vps51

Introduction

Protein transport via vesicular trafficking inside eukary-

otic cells is a highly regulated process that requires

specificity at each transport step. Cargo containing carrier

vesicles derived from a donor membrane must be directed to

0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2005.01.022

$ The novel nucleotide and amino acid sequences reported in this paper

have been submitted to the GenBank TM/EBI with accession numbers

AY444797 (hVps53) and AY444798 (hVps54).

T Corresponding author. Fax: +49 6841 1623092.

E-mail address: stenlie@t-online.de (F. Stenner-Liewen).

their destination before ultimately fusing with an acceptor

membrane. The fusion events are controlled by SNARE and

Rab family proteins.

SNARE proteins are integral membrane proteins located

on vesicle and target organelles that drive membrane

fusion. Rab proteins regulate this process by recruiting

and activating effectors called tethering factors. Tethering

factors are multiprotein complexes that tether designated

membranes before fusion, thus acting upstream of SNARE

proteins. Other than SNARE and Rab proteins, the tether-

ing factors have long withstood any classification

(reviewed by Ref. [1]). Recently, a small region in the

ch 306 (2005) 24–34

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–34 25

amino terminus of these proteins, consisting of two

amphipathic helices that form a coiled-coil motif, was

identified as the common denominator of the tethering

factor family [2].

Among tethering factors, GARP has a role in the

carboxypeptidase (CPY)-containing vesicle transport path-

way [3]. GARP is involved in retrograde transport of

early and late endosomes to the late Golgi. Experiments

in yeast have established that Vps proteins Vps52, 53, 54

form a core complex with a 1:1:1 stoichiometry [3].

Deletion mutants of GARP components in yeast are non-

lethal, but display temperature-sensitive growth impair-

ments, aberrant vacuolar morphology and missorting of

CPY [3]. Knock-out mutants of Vps proteins revealed that

individual components rely on the presence of the other

members for individual stability. The phenotype of single

deletion mutants is indistinguishable from triple deletion

mutant implying an underlying functional complex

(Fig. 1).

Tethering factors are activated by specific regulators to

exert their function. This task is fulfilled by the Ras-like

GTPases of the Ypt/Rab family (for review, see Ref. [4]). In

yeast, GARP is the main effector of Ypt6p, the yeast

homologue of human Rab6 [5].

Yeast strains lacking Ypt6p accumulate transport vesicles

destined for fusion with late Golgi membranes [6,7].

Conversely, overexpression of wildtype or a constitutively

active Rab6 stimulates retrograde transport to the late Golgi

and from the Golgi to the ER in animal cells [8]. These

results indicate an involvement of Rab6 in retrograde

membrane traffic from endosomes to the Golgi and

eventually from the Golgi to the ER. Visualizing experi-

ments in living cells have established that Rab6 governs an

alternate, COPI-independent recycling transport pathway

from Golgi to ER [9].

In yeast, Tlg1p was identified as the SNARE protein

that binds to the Vps complex through its N-terminal

domain [5]. This binding depends on Vps51, making

this small protein the fourth component of the VFT

complex in yeast [10–12]. Like all other members of the

complex, Vps51 possesses a coiled-coil domain. How-

ever, Vps51 is not absolutely required for assembly,

localization, or function of the VFT complex [10–12].

Furthermore, Vps51 shows low conservation and is

possibly absent in higher eukaryotic genomes (own data

and Ref. [10]).

Although the murine and rat homologues of Vps54 have

been cloned [13], none of the human components of the

VFT complex have been described so far. After isolating the

human orthologue of Vps54 when screening a hepatocel-

lular carcinoma cDNA expression library [14], we cloned all

three members of the human Vps complex. This study

reports the characterization of the previously undefined

human VFT complex forming proteins hVps52, hVps53,

hVps54. By analogy to the characteristics of the yeast

homologues, the tight association in a stoichiometric

complex, the typical smooth membrane and Golgi local-

ization, and the interaction with Rab and SNARE proteins,

we refer to this complex as human Golgi associated

retrograde protein complex (hGARP).

Materials and methods

cDNA cloning and plasmids

The following constructs were used in this study: 1a.

Flag-Vps54L full length, 1b. Flag-Vps54L-DC, encoding aa

1–535 1c. Flag-Vps54L-DN carrying aa 634–977 1d. GFP-

SLP-8s (GFP fused to hVps54 short, splicing variant lacking

aa 46–57) 2. hVps52 full length, 3. hVps53 full length, were

each amplified by PCR from clones (2. RZPD clone ID

IMAGp998A1710731Q3, 3. IMAGp998G1912216Q3)

acquired through the RZPD (Deutsches Resourcenzentrum

fuer Genomforschung; www.rzpd.de), Berlin, Germany. After

appropriate restriction digestion, PCR products were inserted

into expression vectors pEGFP-C2 (Clontech, Oceanside,

USA) and pcDNA3 (Invitrogen, CA, USA) using the

respective cloning sites. For the above constructs, the

following forward and reverse primers were used: 1a. 5V-CGGAATTCCGCCACCATGGCTTCAAGCCACAGTTCT-

3Vand 5V-CGCTCGAGTCACCTCTTCTGCTCCCAAAT-3V;1d. 5V-AGTGGGGATCCTATGCAA TGG-3V and 5V-TTGTGGATCCGGTCCAAATCTTTAAGCCTTT-3V; 2. 5V-CGGCTCGAGATGGCCGCCGCTGC-3Vand 5V-CGGC-TCGAGTCAGAAGTTGGGCTTATGCTT-3V. 3. 5V-CGGA-ATTCATGATGGAGGAGGAGGA-3Vand 5V-CGGCTCGA-GCAGGGGACATC ATCAAT-3V. 1b and 1c were con-

structed by subcloning fragments using internal XbaI and

EcoRI sites of hVps54L (long form).

Human Syntaxin 6, Syntaxin 10, and Rab6b were

cloned from a cDNA expression library made from testis

using the following primer pairs: Syntaxin 6: 5V-CGGGATCCATGTCCATGGAGGACCC-3V and 5V-GCTCTAGATCACAGCACTAGG AAGAGG-3V, Syntaxin

10: 5V-CGGGATCCACTGACATGTCTCTCGAAGA-3V and

5V-GC TCTAGATCAGAGAGAGAATAGTAAGATGAGA-

3V, Rab6b: 5V-CGGGATCCATGTCC GCAGGGGGAGAT-

TTTG-3V and 5V-GCTCTAGATTAGCAGGAGCAGCCGC-CCT-3V to amplify the full length cDNAs and inserted into

expression vectors pEGFP-C2 (Clontech, Oceanside, USA)

and E2 (Abcam, Cambridge, UK), utilizing the in-frame

BamHI and the XbaI restriction sites. Rab6T27N, a Rab6

mutant with strongly reduced GTP binding affinity [15],

was created by introducing the modified sequence (T27N)

with corresponding primers using the QUICKCHANGE-kit

(Invitrogen). To facilitate subcloning of hVps54 full length

into different expression vectors, the naturally occurring

EcoRI site at position bp 1905 was abolished by site

directed mutagenesis to create a silent mutation of C to T.

The integrity of all constructs was confirmed by sequencing,

restriction digest, and Western blotting. Some constructs

Fig. 1. Predicted structural domains and comparison of GARP complex proteins in human and yeast. (A) A schematic representation of the conserved tethering

factor complex domains (TFCD, white boxes) of the human Vps proteins and their relative position (amino acids) within the proteins. (B) Coiled-coil domains

in human (left) and yeast (right) Vps proteins. Predictions were made with COILS version 2.1 at the expasy server, using Lupas algorithm [29] MTIDK matrix,

window length 28 residues and weighting of hydrophobic residues. x-axis represents aa residues, y-axis contains the probability of coiled-coil-forming. Identity

and similarity values were calculated using MatGAT 2.0 program by Ledion Bitincka.

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–3426

were only partially sequenced, but all novel genes were

confirmed by full sequencing. For RT-PCR analysis of

alternative transcripts, the following primers were used: 5V-TTTAATGCTGCTGGGAGATTTACTTT-3V and 5V-TTGAGTGGTGATTTTATGCAA TGGC-3V. The primers

were selected in exon 1 and exon 3 flanking the

alternatively spliced coding region of exon 2. This primer

combination yielded a 253 and 217 bp product for the long

and short isoforms, respectively. Contaminating genomic

DNA would yield a N2 kb product. cDNAs were generated

from tissues as described previously [14]. The PCR

conditions following the reverse transcription procedure

were: 958C for 5 min, 558C for 30 s, 728C for 20 s, 30

cycles.

Fig. 2. Expression of hVps54 in tissues and cell lines. (A) First-strand

cDNAs generated from various mRNAs were used as templates and

amplified with primers flanking Exon2 (aa 46–57), thus testing for

abundancy of alternative transcripts hVps54L and hVps54s. PCR products

were analyzed by agarose gel electrophoresis and visualized by UV

illumination of ethidium bromide-stained gels. Lane 1, testis; lane 2,

hepatocellular carcinoma/HCC-1; lane 3, HCC-2; lane 4, diluted plasmid

pos. control carrying hVps54 short form; lane 5, kidney; lane 6, skin/

melanoma; lane 7, rectum carcinoma; lane 8, lymph node; lane 9, spleen;

lane 10, liver; lane 11, negative control reaction performed without cDNA.

The PCR products vary in length by 36 base pairs (253bp and 217bp). 2

different molecular weight markers were used. Lower panels B and C show

characterization of the antibodies generated for this study. (B) Total protein

lysates (20 Ag) of HEK 293 cells native and transfected with various

constructs used in this study. (Left side) Anti-Vps54-peptide antibody

detecting endogenous hVps54 and tagged fusion hVps54 proteins. Lane 1,

native HEK 293 cells; lane 2, GFP-Vps54s, the shifted second band

corresponds to the GFP-fusion protein; lane 3, Flag-hVps54-D-C (aa 1–

637); lane 4, Flag-hVps54-D-N (aa 634–977); only endogenous hVps54 is

detected by anti-hVps54; lane 5, Flag-hVps54 full length. (Right side) a-

Flag-antibody detecting respective fusion proteins. Lane 6, Flag-hVps54-D-

C; lane 7, Flag-hVps54-D-N; lane 8, Flag-Vps54 full length. Arrowhead (N)

indicates position of endogenous hVps54 band. (C) Total protein lysates

(20 Ag) of HEK 293 cells native (lane 1 and 3) or transfected with 2 Agplasmid encoding either Myc-tagged-hVps52 (lane 3) or HA-tagged

hVps53 (lane 4). Arrowhead (N) indicates position of specific bands,

*marks a background band detected by anti-hVps53 antibody.

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–34 27

Cell culture and transfections

Cell lines used were obtained commercially from ATCC

and maintained at 378C, 95% humidity, and 5% CO2. HEK

293 cells were propagated in DMEM (PAA, Pasching,

Austria), A549 cells in Ham’s (PAA), and Cos7 cells in

RPMI media (PAA). All media were supplemented with

10% FCS, 50 IU/ml penicillin/streptomycin and glutamine

(Gibco Invitrogen, Karlsruhe, Germany). DNA transfec-

tions were performed using PerFectin (Peqlab Biotechno-

logie, Erlangen, Germany) according to the manufacturer’s

protocol.

Cell fractionation

Subcellular fractions of dog pancreas were prepared as

described elsewhere [16]. In brief, cytosol fraction was

isolated from the top and the ribosome free sER/Golgi

fraction from the interphase of a 1.3 M sucrose cushion after

sedimentation centrifugation (140,000 gav, Beckman Ti

50.2 rotor, for 2.5 h). [17]. Fractions were tested for cell

compartment specific proteins by immunoblotting (GAPDH

for cytosol, p58 for sER/Golgi).

Antibodies and immunoblot analysis

Antibodies used in this study were: anti-HA antibody

(Roche, Mannheim, Germany), anti-Flag antibody (Sigma-

Aldrich, Taufkirchen, Germany), anti-E2 antibody (Abcam),

anti-myc antibody (clone 9E10, Zymed, San Francisco,

USA), monoclonal anti-Golgi 58K protein marker (Sigma-

Aldrich), monoclonal Golgi marker GM130 (BD Bioscien-

ces, NJ, USA), monoclonal anti-human transferrin Receptor

(Molecular Probes), monoclonal anti mannose-6-phosphate

receptor (Calbiochem, CA, USA), and monoclonal anti-

GAPDH antibody (Abcam).

Antibody generation

For preparation of polyclonal antibodies against hVps52,

hVps53, hVps54 antisera were generated by repeated

immunization of rabbits with 15-mer synthetic peptides.

Prior to injection, the peptides corresponding to sequences

located within the respective hVps proteins (peptide for

hVps52 aa 79–93, for hVps53 aa 61–75, and for hVps54 aa

138–152) were synthesized, conjugated to keyhole limpet

hemocyanin, and emulsified in complete Freund’s adjuvant.

To demonstrate the specificity of these antibodies, total

protein lysates from HEK 293 cells transiently transfected

with different hVps constructs (as positive controls) were

prepared, normalized for total protein content (20 Ag), andsize-fractionated in a 12% polyacrylamide gel under stand-

ard SDS–PAGE conditions. Proteins were then transferred

onto PVDF membranes (Millipore, Bedford, USA) and

incubated with a 1:1000 (v/v) dilution of anti-hVps antisera

and subsequently incubated with peroxidase-conjugated

goat-anti-rabbit-antibody (Bio-Rad, Munich, Germany).

Detection was accomplished by an enhanced chemilumi-

nescence system (ECL, Amersham Biosciences, Freiburg,

Germany).

Co-immunoprecipitation

HEK 293 cells were co-transfected with a total of 2 Ag of

plasmid DNA. 48 h after transfection, HEK 293 cells were

collected, suspended in lysis buffer (20 mM Tris–HCl pH

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–3428

7.4, 150 mM NaCl, 1% Triton X-100, 10% Glycerol, and

complete protein inhibitors (Roche, Mannheim, Germany)),

and lysed by repeated aspiration through a 27 gauge needle.

Extracts were preabsorbed on recombinant Protein G-

Sepharose (Zymed) in wash buffer (lysis buffer without

TritonX-100) and rotated overnight at 48C with 20 Alsepharose and 2 Ag antibody. Beads were then washed 6

times in wash buffer. Bound proteins were recovered by

boiling the beads for 5 min in protein sample buffer [18],

loaded onto SDS–polyacrylamide gels, and transferred to

PVDF membranes (Millipore). Proteins were detected using

anti-HA antibody, anti-Flag antibody, anti-E2 antibody, or

anti-myc-antibody followed by secondary antibody and

ECL as above. For the Syntaxin precipitation experiments,

a cross-linking reagent, 1 mM DSP was added for 30 min at

48C. After quenching the reaction by 15 min incubation in

the presence of 50 mM Tris, pH 7.5, the buffer was

readjusted to lysis buffer conditions. Immunoprecipitation

was done as described above.

Co-immunoprecipitation of endogenous Vps proteins

Untransfected HEK 293 cells were lysed, washed, and

preabsorbed as above. Then extracts were preabsorbed on

recombinant Protein G-Sepharose (Zymed) in wash buffer

(lysis buffer without TritonX-100) and rotated overnight at

48C in the presence of the relevant antibody. Recovery,

SDS–PAGE separation, and transfer to PVDF membranes

were described previously. Protein detection was performed

with polyclonal sera again followed by secondary antibody

and ECL development.

Immunofluorescence microscopy

Cos7 or A549 cells (105) were seeded into 4-well,

covered chamber slides (Nalge Nunc, USA). In some cases,

cells were transfected with LipofectAMINE (Life Technol-

ogies, Inc., USA) with indicated plasmids (0.4 Ag DNA) andincubated at 378C for 24 h. Thereafter cells were fixed and

permeabilized with either 1:1 (v/v) methanol/acetone on ice

for 5 min or alternatively with 4% paraformaldehyde for 20

min followed by 5 min 0.2% saponin treatment at room

Fig. 3. GARP associations. (A) HEK 293 cells were transfected with 1 Ag of eac

prepared and subjected to immunoprecipitation (IP) with either anti-Myc (left

complexes were then analyzed by SDS–PAGE followed by immunoblotting (WB

Alternatively, 10% of the input lysates were run directly on gels (20 Ag of protein) aexpression of proteins. Unrelated proteins carrying the same epitope-tag (survivin

and subjected to immunoprecipitation as in A. For precipitation, anti-hVps54 (upp

hVps52 was detected by its polyclonal antiserum. Anti-myc antibody served as

hVps52 was the positive control. (C) HEK 293 cells were transfected as in A. The h

E2-tagged-Syntaxin 6 (Stx 6). Western blotting (middle and lower panels) of lysa

HEK 293 cells were transfected and subsequently co-immunoprecipitated as in A. h

Rab6 T27N mutant (mut) that has a strongly reduced affinity for GTP and is thu

transfected/co-immunoprecipitated as in A. Because Syntaxin 6 did not precipita

Detection of hVps proteins and marker proteins in preparations of pancreas cytosol

separated in an SDS polyacrylamide gel, transferred onto PVDF membrane, and

enhanced chemiluminescence. Golgi preparation was confirmed by detection of 5

temperature. The slides were then extensively washed and

preblocked with phosphate-buffered saline (PBS) containing

10% BSA and 1% normal goat serum. Primary antibodies

were typically applied at dilutions of 1:100 to 1:200 for 12 h

at 48C. After extensive washing with PBS, secondary

antibodies, Alexa Fluor 488 goat anti-mouse, and Alexa

Fluor 594 goat anti-rabbit (Molecular Probes) were used at a

dilution of 1:400 for 20 min. After another three washing

steps, gaskets were removed and the slides sealed using

fluorescence preserving mounting medium (MobiGLOW,

MoBiTech, Goettingen, Germany). In the case of Fig. 5B,

incubation was performed in two steps. After methanol/

aceton fixation and preblocking as before, slides were

probed with antihuman Transferrin Receptor (Molecular

Probes) for 2 h. After extensive washing, this was followed

by incubation with secondary antibody, Alexa Fluor 488

goat anti-mouse. Then the slides were fixed with 4%

paraformaldehyde for 20 min. The slides were then

incubated for 1 h with prelabeled anti-E2 antibody (Zenon

Alexa Fluor 594 mouse IgG1 labeling kit, Molecular

Probes). Slides were sealed after three washing steps using

fluorescence preserving mounting medium as before.

Confocal microscopy (Figs. 4A–I and 5A, B) was

performed using a two-photon system (MRC 1024,

Bio-Rad).

Results

Identification and cloning of SLP-8, the human homologue

of Vps54p, and its complex partners

Using autologous serum from a patient with hepatocel-

lular carcinoma, we screened a cDNA expression library

generated from that patient’s tumor tissue [14]. We obtained

a C-terminal fragment and cloned the full open reading

frame coding for a 110-kDa protein by a cDNA race

strategy. The sequence was deposited in GenBank (acces-

sion no. AF102177). The clone from the HCC library was

found to be mutated at bp 2884 (loss of a C) leading to a

frame shift and alteration of the C-terminus of the protein.

The wildtype nucleotide and amino acid sequence was

h epitope-tagged plasmid as indicated (lane1). After 48 h, cell lysates were

panel) or anti-Flag antibody (middle, right panel). The resulting immune

) using either anti-HA (left and right panel) or anti-Myc antibody (middle)

nd analyzed by immunoblotting (WB) using indicated antibodies to confirm

and Traf-6) served as controls. (B) Untransfected HEK 293 cells were lysed

er panel) and anti-hVps53 (lower panel) antibodies were used. Endogenous

an unrelated negative control. Immunoprecipitation of hVps52 with anti

Vps proteins were tested for their ability to interact with E2-tagged Rab6 o

tes assuring proper expression of the respective proteins in the assays. (D

Vps52 protein was tested for differences in binding Rab6 wildtype (wt) or a

s preferentially in the GDP-bound conformation. (E) HEK 293 cells were

te hVps52 in C, it was used as a negative control in this experiment. (F

and sER/Golgi of canine origin. 10 Ag of the two different preparations wasprobed with the antibodies indicated. Finally, proteins were detected by

8 kDa Golgi protein. GAPDH served as a cytosol marker.

.

-

r

)

)

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–34 29

retrieved and confirmed using cDNAs generated from

different cell lines and normal tissues (accession no.

AY444798). Initially, the novel protein was named SLP-8.

Database and domain searches revealed SLP-8 to be a

protein with a predominantly helical fold bearing a short

coiled-coil domain, but no evident function was attributable.

The yeast homologue of SLP-8 was found independently by

several groups and is referred to as LUV1/Rki1p/Tcs3p/

Vps54p [19]. The systematic term Vps54p has prevailed and

for clarity we decided to refer to SLP-8 and its complex

partner proteins as human Vps proteins, e.g., hVps54,

hVps52, and hVps53. The latter two were identified using

the yeast homologues as bait for iterative Blast searches in

public databases [20].

A noteworthy fact is the existence of one shorter isoform

of each of the three hVps proteins. The isoforms originate

through alternatively spliced transcripts lacking one exon of

variable length (amino acids (aa) 313–374 in hVps52s, aa

96–124 in hVps53s, and aa 46–57 in hVps54s). For

hVps54s, we confirmed the presence of a long and short

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–3430

form by RT-PCR (Fig. 2A). As for the long and short

isoforms of hVps52 and hVps53, several ESTs for both

forms exist in public databases. The shorter isoform of

hVps52 lacks a significant portion of the coiled-coil domain.

Deletion of the coiled-coil domain in yeast homologues

leads to an inability to complement the growth of the

respective deletion mutants and loss of association with the

remaining VFT complex [10].

Biological computational results

A comparison of the components of GARP complexes in

different species displays some significant differences. The

orthologues of hVps54 in Caenorhabditis elegans

(T21C9.2) and Drosophila (scat) possess a N-terminal

zinc-finger domain [21] that is absent in the yeast and

human counterparts. Vps51, the fourth component of the

GARP in yeast, appears to have no homologue in higher

eukaryotic cells. Blast searches indicate that this protein

may not be conserved in higher eukaryotic genomes (own

searches and Ref. [10]).

Gene targeting experiments in yeast show that VFT

genes are non-essential but cause reduced growth in a

temperature-sensitive manner [3]. Drosophila mutants

lacking Vps54/scat are viable, but males are infertile

due to immobile spermatozoa [22]. Gene targeting experi-

ments using RNAi against CED Vps54 have been

published in the worm database (www.wormbase.org,

release WS111, Oct. 2003), but are inconclusive. While

one group (Maeda I. and colleagues) found gonad abnor-

malities, another group (Ashrafi K. and colleagues)

observed a wildtype phenotype. RNAi experiments spe-

cific to C. elegans Vps52 and Vps53 orthologues have

yielded no phenotype.

Secondary structure predictions done for human Vps

proteins with Jpred [23] reveal predominantly helical folds.

By computational analysis, GARP proteins are distantly

related to other tethering factors, the COG and the exocyst

by a short helical N-terminal domain [2,21].

Interaction and subcellular distribution of the hVps proteins

To examine the conservation of the VFT complex further,

the human components were tested independently for their

ability to interact by co-immunoprecipitation assays. As

shown in Fig. 3A, each of the three members interacted with

each other. To verify the results, endogenous complex

partners in HEK 293 cells were precipitated using the

generated antibodies against hVps proteins. Endogenous

hVps52 could be immunoprecipitated by either hVps54 or

hVps53, but not by an unrelated anti-myc antibody (Fig.

3B). Changing orientation of precipitation partners did not

alter the result (data not shown).

We next asked whether human GARP is an effector of

the small GTPase Rab6. As shown in Fig. 3C, E2-tagged

Rab6 specifically precipitated hVps52, but no other member

of the VFT complex. Having determined this interaction, we

examined whether Syntaxin 6, the putative human homo-

logue of TLG1p, associates with members of the GARP. No

binding of any hVps proteins with this SNARE protein

could be detected, even in the presence of crosslinking

reagents (Fig. 3C). Extending our search for an appropriate

interacting SNARE protein for the human GARP, we next

tested Syntaxin 10. For this SNARE protein, a tissue-

restricted expression has been reported [24]. However,

expressed sequence tags (ESTs) in public databases of a

shorter isoform of Syntaxin 10 (Supplementary Fig. 1)

derived from virtually every tissue indicate an ubiquitous

expression. In this study, the short isoform of Syntaxin 10

was used. In co-immunoprecipitation experiments, Syntaxin

10 precipitated endogenous hVps52 (Fig. 3E).

At the subcellular level, human VFT proteins localize in

a typical vesicular pattern (Fig. 4). When compared to Golgi

markers, e.g., GM130, the hVps protein signals are more

dispersed throughout the cell but accumulate and overlap

with GM130 in a polarized perinuclear area (Figs. 4A–C).

Likewise, the late endosome marker mannose-6-phosphat

receptor (MPR) and the early endosome marker transferrin

receptor overlap in co-staining experiments with Vps

proteins at the perinuclear envelope. In the peripheral

cytoplasm, however, no identical staining is observed (Figs.

4J–L and not shown).

Subcellular fractionation experiments show that Vps52,

Vps53, and Vps54 are detected by their respective anti-

bodies in the smooth membrane/Golgi fraction but not in the

cytosol (Fig. 3F).

Having determined that Rab6 is an interaction partner of

the human GARP, confocal microscopy was applied to

assess this result in vivo. Endogenous hVps52 co-localized

with Rab6 in cells expressing an E2-tagged Rab6 protein

(Fig. 5A).

Finally, we examined the localization of Syntaxin 10 in

conjunction with the VFT protein hVps52 by confocal

microscopy. Syntaxin 10 moderately expressed as an E2-

fusion protein was found in vesicles near the Golgi and co-

localized with endogenous hVps52 at inner vesicles (Fig.

5B). Overexpression of Syntaxin 10 led to the formation of

giant perinuclear structures. Co-staining experiments

revealed these structures to be positive for hVps52 (Fig.

5C), indicating a recruitment or a prolonged binding of this

tethering factor to Syntaxin 10. Further staining experiments

showed that these structures contain transferrin receptor

(Fig. 5D), indicating an endosomal origin. Syntaxin 6 did

not co-localize with Vps52 and overexpressing Syntaxin 6

did not result in the formation of giant endosomes (data not

shown).

Discussion

In this study, the human GARP complex could be defined

by showing that the human homologue of Vps54p forms a

Fig. 4. Subcellular distribution of hVps proteins. After fixation with methanol/acetone, COS7 cells were incubated with a-hVps52 (A–C), a-hVps53 (D–F, J–

L), and a-hVps54 (G–I). Then respective secondary antibodies were applied (A, D, G, J, Alexa Fluor 594 goat anti-rabbit, red). Counter-staining was done with

Golgi marker GM130 (A–I) and late endosome marker anti-mannose-6-phosphat-receptor (J–L) (secondary antibody Alexa Fluor 488 goat anti-mouse, green).

Right side panels show merged images. The inset in L represents a magnification of the cells’ perinuclear zone. (For interpretation of the references to colour in

this figure legend, the reader is referred to the web version of this article.)

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–34 31

trimeric complex with human Vps52 and human Vps53. The

GARP proteins in humans each possess 2 isoforms. At this

point, no differences in binding affinities between long and

short isoforms were seen and no significant difference in

intracellular expression levels of either isoform was deter-

mined. The human Vps proteins have a predicted predom-

inant helical fold and contain coiled-coil domains. These

features are conserved among VFT complex forming

proteins from unicellular to multicellular organisms. A N-

terminal zinc-finger-like domain of Vps54 found in Droso-

phila and C. elegans is not conserved in mammals,

indicating an acquisition of functional differences of GARP

during evolution. In comparison to Saccharomyces cerevi-

siae, in higher eukaryotic organisms GARP seems to

encompass three members instead of four. The recently

described fourth component of GARP in yeast Vps51 [10–

12] seems to have no evident mammalian homologue ([10]

and own searches). However, in yeast, GARP is a stable

trimeric complex without Vps51 [10–12]. Conversely,

intracellular levels of Vps51 are not dependent on the

presence of the core GARP complex. These observations

have led to the conclusion that Vps51 is not of structural

significance in the assembly of the complex [12].

A model of Vps51’s regulatory role in tethering events

has been proposed by Conibear and Stevens [12]. Rather

than being an essential component of GARP, Vps51 could

be a soluble cytosolic factor. By interacting with TLG1p,

Vps51 stabilizes the t-SNARE in an open conformation

thereby activating SNARE assembly and driving membrane

fusion. In this scenario, Vps51 would be dispensable for

Fig. 5. Investigation of in vivo association of hVps proteins with Rab6 and Syntaxin 10. (A) A549 cells were transfected with E2-Rab6, fixed with 4%

paraformaldehyde, and then labeled with anti-E2 and anti-Vps52. This was followed by incubation with goat anti-mouse (green, A) and goat anti-rabbit (red,

B), panel C merged. In D–F, A549 cells were transfected with E2-Syntaxin 10, fixed at 24 h or earlier with 4% paraformaldehyde, and subsequently labeled

with anti-E2 (A, secondary antibody goat anti-mouse green) and anti-Vps52 (B, secondary antibody goat anti-rabbit red), panel C merged. Investigation of

Syntaxin 10 overexpression. (B, panels A–C) A549 cells were transfected with E2-Syntaxin 10, fixed at 36 h to promote overexpressing of Syntaxin 10.

Fixation was done as in A. (Panel A, secondary antibody goat anti-mouse green) and anti-Vps52 (panel B, secondary antibody goat anti-rabbit red), panel C

merged. dBlob-likeT overlapping structures were observed perinuclear. (Panels D–F) COS7 cells were transfected as in C. Cells were fixed with methanol/

acetone and labeling was done in subsequent steps as described in Materials and methods. Syntaxin 10 was detected with prelabeled anti-E2 antibody (panel D,

red). Transferrin receptor was detected with anti-Transferrin-antibody (panel E) followed by secondary antibody (goat anti-mouse, green). (Panel F) Formation

of giant endosomes positive for Syntaxin10 and transferrin receptor. (For interpretation of the references to colour in this figure legend, the reader is referred to

the web version of this article.)

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–3432

GARP and its function until the actual membrane fusion

event takes place. Then Vps51 would make a brief

appearance as a regulator. According to this hypothesis, an

explanation for the absence of Vps51 in higher eukaryotic

cells could be a functional substitution of this regulator by

another protein in the vicinity of the Golgi. But the

existence of a human homologue of Vps51 or its substitute

might not to be determinable by computational means and

will have to await further investigations.

Rab-proteins, conductors of vesicle transport, have

expanded significantly from yeast (11 members) to C.

elegans (29 members) to humans (N60 members) [25]. The

abundance of human Rabs seems to reflect the increasing

complexity and specialization of targeting vesicles between

organelles in the course of evolution. The recruitment of the

Vps complex by Ypt6p to late Golgi-membranes in yeast is

mirrored by the herein described interaction of Rab6 and

hVps52 in mammalian cells. The latter binding is energy

H. Liewen et al. / Experimental Cell Research 306 (2005) 24–34 33

dependent, thus the GTP-activated form of Rab6 displays a

binding preference to Vps52. This supports the idea that the

human GARP is an effector of the Rab6 protein and that it

has a phylogenetically conserved role in vesicle transport

events.

In contrast to the Rab protein family, the number of

SNARE proteins has remained mostly unchanged in yeast,

flies, and worms, and has only modestly increased in

humans (from 21 to 35 members [25]). It is speculated

that the principle SNARE machinery has not been

modified, instead tissue-specific expression of different

SNAREs has been added to expand specificity. In case of

human GARP, Syntaxin 10 instead of Syntaxin 6 seems to

have adopted the role of Tlg1p. However, if a human

homologue of Vps51 exists, Syntaxin 6 could still play a

co-regulatory role in the docking process. Given the

economy of nature when it comes to SNARE proteins, it

is surprising that retrograde vesicle transport in humans

was equipped with an innate SNARE protein. The

association of GARP with Syntaxin 10 is an example

for functional diversification during the evolution of

SNARE proteins in humans.

Remarkable is the formation of giant endosomes follow-

ing overexpression of Syntaxin 10. Gross disturbance of

Syntaxin 10 levels leads to loss of this SNARE’s ability to

promote transient endosome fusion. Duclos et al. have

shown that disruption of Rab5’s regulatory function leads to

a strikingly similar phenotype [26]. These observations

demonstrate that traffic of endosomes is a delicately timed,

well balanced, and tightly regulated process.

The present understanding of mammalian endocytic

recycling pathways is incomplete due to the abundance

and complexity of the molecules involved. Human Vps

proteins’ restricted perinuclear co-localization with man-

nose-6-phosphate receptor and transferrin receptor is a

noteworthy finding. It implies that Vps proteins mark

distinct endosomes that meet early and late endosomes

most likely at the endocytic recycling compartment (ERC)

(reviewed in [27]). Passage of VFT complex through the

ERC is supported by the established role of its interaction

partner Rab6 in the endosome/TGN recycling pathway [28].

Future work elucidating the intracellular routes of the

VFT-positive endosomes and their contents will add to our

knowledge of vesicle transport.

Acknowledgments

We are indebted to Gregor Reither and Carsten Ehrhardt

for generous assistance with confocal microscopy, and Juan

M. Zapata for fruitful discussions and scientific advice. We

thank Wolfgang Nastainczyk for help with antibody gen-

eration against hVps52 and hVps53, and Thomas Bauknecht

(Lilly, Germany) for support with the generation of the

hVps54 antibody. Finally, we acknowledge Cora Stefan’s

excellent technical assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.yexcr.2005.

01.022.

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