Distinct Transport Vesicles Mediate the Delivery of Plasma Membrane Proteins to the Apical and ...

14 Pages·2002·2.98 MB·English

Distinct Transport Vesicles Mediate the Delivery of Plasma Membrane
Proteins to the Apical and Basolateral Domains of MDCK Cells
Angela ,sseN-regnidnaW Mark K. Bennett, Claude Antony, and iaK Simons
Cell Biology Program, European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, Federal Republic of Germany
Abstract. Immunoisolation techniques have led to the glycoproteins permitted the resolution of apical and
purification of apical and basolateral transport vesicles basolateral vesicle fractions. The immunoisolated vesi-
that mediate the delivery of proteins from the trans- cles contained a subset of the proteins present in the
Golgi network to the two plasma membrane domains starting fraction. Many of the proteins were sialylated
of MDCK cells. We showed previously that these as expected for proteins existing the trans-Golgi net-
D
o
transport vesicles can be formed and released in the work. The two populations of vesicles contained a w
n
lo
presence of ATP from mechanically perforated cells number of proteins in common, as well as components ad
e
(Bennett, M. K., A. Wandinger-Ness, and K. Simons, which were enriched up to 38-fold in one fraction rel- d fro
1988. EMBO (Euro. Mol. Biol. Organ.) J. ative to the other. Among the unique components, a m h
7mo:n4i0t7o5r-e4d0 85). the purifiUcsaitnigo n virally of infectetdh e trans-Golgi cells, we derived have usniungm ber Triton of transmembrane X-114 phase partitioning. proteins could This be work identified pro- ttp://rup
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vesicles by following influenza hemagglutinin or vesic- vides evidence that two distinct classes of vesicles are ss.o
ular stomatitis virus (VSV) G protein as apical and responsible for apical and basolateral protein delivery. rg/jcb
bgarsaodliaetnetr al centrifugation markers, respectively. revealed that Equilibrium hemagglutinin density vColovmedm on in prvoetseicilne budcdoimnpg onents and are fusion suggested steps, whilteo be in- /article
-p
containing vesicles had a slightly lower density than unique components may be required for specific recog- df/1
1
those containing VSV-G protein, indicating that the nition events such as those involved in protein sorting 1/3
/9
two fractions were distinct. Antibodies directed against and vesicle targeting. 87
/1
the cytoplasmically exposed domains of the viral spike 06
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1
7
4
/9
8
7
.p
d
S LAREVE classes of proteins including lysosomal en- have been used extensively as a model system to study pro- f by g
u
zreygmuelsa,t ed plasma secretion membrane follow a proteins, common routand e proteins from their of tein Newly sorting synthesized in epithelial plasma cells membrane (McRoberts proteins et al., have 1981). been est on
1
site of synthesis in the ER through the cisternae of the Golgi shown to be sorted intracellularly using MDCK cells in- 4 Ja
complex. Upon arriving in the trans-Golgi network (TGN) t fected with enveloped RNA viruses (Matlin and Simons, nu
a
the proteins in transit must be sorted for delivery to their 1984; Misek et al., 1984; Rindler et al., 1985; Pfeiffer et al., ry 2
0
final destinations (for reviews see Griffiths and Simons, 1985). The spike glycoproteins of such viruses follow the 23
1986; Klausner, 1989). In epithelial cells, the plasma mem- same intracellular transport pathway as endogenous plasma
brane consists of two domains each with a distinct protein membrane proteins and are delivered differentially to the two
composition (for review see Simons and Fuller, 1985; Rod- domains without requiring additional virally encoded prod-
riguez-Boulan and Nelson, 1989). This necessitates an addi- ucts (Rindler et al., 1984; Fuller et al., 1985; Jones et al.,
tional sorting step for the segregation of apical from baso- 1985; Caplan et al., 1986; Gottlieb et al., 1986b; Stephens
lateral plasma membrane proteins. et al., 1986). Influenza hemagglutin (HA) is transported to
MDCK ceils form polarized monolayers in culture and the apical domain, while vesicular stomatitis virus (VSV) G
protein is delivered to the basolateral domain (Rodriguez-
Boulan and Pendergast, 1980). The advantages afforded by
Mark Bennett's present address is Department of Biological Sciences, Stan-
using the viral glycoproteins as model apical and basolateral
ford University, Stanford, CA 94305-5020.
Claude Antony's present address is Institut Jacques-Monod, Physiolo- plasma membrane proteins are several fold. The proteins are
gique du D~veloppemant, Universit6 de Pads VII-CNRS, Tour 43, 2 Place highly expressed during viral infection and can be detected
Jussieu, F-75005 Pads, France. readily. Because host protein synthesis is inhibited by viral
infection, the viral glycoproteins represent the sole proteins
.1 Abbreviations used in this paper: HA, hemagglutin; NSE N-ethylmalei-
in transit. However, even in the absence of host protein syn-
mide-sensitive factor; TGN, trans-Golgi network; VSV, vesicular stomati-
tis virus. thesis, viral protein sorting continues for several hours after
© The Rockefeller University Press, 0021-9525/90/09/987/14 $2.00
The Journal of Cell Biology, Volume 111, September 1990 987-1000 987
infection (Rodriguez-Boulan and Pendergast, 1980; Fuller et methionine-free E-MEM containing 01 mM Hepes, pH 7.3, 0.2% (wt/vol)
al., 1984). This suggests that the proteins involved in sorting BSA, and 0.35 g/l sodium bicarbonate (instead of the usual 2.2 g/l). Water
and proper delivery must be reused during multiple rounds bath medium was E-MEM supplemented with 01 mM Hepes, pH 7.3, 0.2%
BSA, 0.35 1/g sodium bicarbonate, 20 ~g/ml cyclobeximide, and 150 mg/l un-
of transport.
labeled methionine (10-fold excess). Metabolic labeling medium consisted of
It is thought that apical and basolateral plasma membrane E-MEM containing 01 mM Hepes, pH 7.3, 2.5% FCS, and 5.1 mg/l unla-
proteins are sorted as they emerge from the TGN by inclu- beled methionine (1/10th the normal concentration).
sion into separate vesicular carriers which are then targeted
to the appropriate membrane domain. In vivo these transport
Cell Culture
vesicles are transient intermediates and not very abundant.
Therefore, we have developed an in vitro system that allows MDCK strain II cells were grown and passaged as described previously (Mat-
the isolation of TGN-derived transport vesicles with the aim lin et al., 1981). The cells from one confluent 75-cm 2 flask were seeded on
six 24-mm diameter, 0.4-/zm pore size, premounted Trauswell polycarbonate
of identifying the molecular machinery involved in protein
filters as described (Bennett et al., 1988). Alternatively, for large scale isola-
sorting and vesicle targeting (Bennett et al., 1988). MDCK
tion of vesicles the cells from one flask were ,er suspended in 20 ml of growth
cells were grown on permeable filter supports allowing them medium and seeded on a single 100-mm diameter, 0.4-#m pore size Tmnswell
to attach tightly to the substrate and to form a fully polarized filter. The filters were ttmmferred to special holders in a Petri dish containing
monolayer. The cells were then infected for short times with 140 ml of growth medium. All filter cultures ware maintained at 37°C and
5 % CO2 for 2-3 d. The Transwell filters were a kind girl from Hank Lane,
influenza or VSV and incubated at 20°C causing the trans-
Costar (Cambridge, MA).
port markers to accumulate in the TGN (Marlin and Simons,
1983; Grifliths et al., 1985; Hughson et al., 1988). Subse-
Viral Infection
quently, the cells were mechanically perforated with the aid
of a nitrocellulose filter. This introduced holes in the plasma Fowl plague virus and VSV stocks were prepared as described (Bennett et D
o
membranes but left the cells attached to the filter support "(Hughsoan l., 1988). et Influenza al., 1988). PR8 Infections and WSN were ts61 performed stocks were by prrienpsairnegd the as filters described with wnloa
with their subcellular organization intact (Bennett et al., d
infection medium and transferring small (24-mm) filters to six-well dishes ed
1989). Using this system, we demonstrated previously that and large (100-mm) filters to 10-cm dishes. The virus stocks were diluted fro
m
HA and VSV-G protein, accumulated in the TGN before per- in infection medium and added to the apical side of the filters, 100 1# per h
mfeomrabtriaonn,e were vesicles released (Bennett from et the al., perforated 1988). The cells vesicles in sealed had 2vi4r-umsm and filter PP,8 and used 1 ml was per 20 100-mm pfu/cell, filter. of WSN The concetntsrattioln 4 pfu/cell, of and fowl of plague VSV ttp://rup
50 pfu/cell. The viruses were allowed to adsorb to the cells for 1 h at 37°C re
the expected topology for authentic transport vesicles and re- (310C used for WSN ts61), the inoculum was removed and fresh infection ss.o
qmueimrberda nes ATP fforro m their such formation perforated and cells release. was Thquei te release specific: of pmeerd ium 100-mm was filtear dded was to baodtdhe d sides to the of apical the filter. side 1 and ml 2.5 per ml 24-ram per 24-mm and 01 and ml rg/jcb/a
oernsl, y or low ER-derived levels of residveesnitcl es Gweorleg i recovered markers, in endocytic the incubation mark- we 2r1e ml continued per 100-mm for an filter additional was added 2.5 h to at the 37°C basolateral (fowl #ague side. virus, The infectionPRS, s and rticle-p
medium (Bennett et al., 1988). The TGN-derived transport VSwaVs) replaced or 3.5 h with at 39"C water (WSN bath mediumts61) in a5n%d tCO2. he infected Subsequently, cells were the incubated medium df/111
vesicles have now been purified for further characterization for 2 h in a water bath at 20°C. /3/9
8
and resolved into apical and basolateral fractions. 7
/1
Radioactive Labeling 06
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Materials and Methods Pulse labeling of infected cells was done with 24-mm filters just before the 4/9
8
20°C incubation as described (Bennett et al., 1988). Metabolic labeling was 7.p
Materials done using 100-mm filters as follows. 2-d-old filter cultures were transferred df b
to 10-cm dishes and washed twice with metabolic labeling medium at 370C. y g
gMeendbieai m, and FRG) reagents and for Biochrom cell culture (Berlin, were FRG). purchased Creatine from phosphate Gibco Biocult and crea- (Eg- and10 ml 21 metabolic ml of the labeling same memdeiduimu m was containing added to 1 the mCi apical 35Smethionine side of each filter was uest o
n
tine kinase were obtained from Boehringer Mannheim (FRG); immobilized added to the basal side. The filters were incubated for 21 h at 37°C, 5% 14
papain from Pierce (Rodgan, FRG); and purified mouse IgG, ATP, BSA, CO:. For subsequent viral infection the apical medium was aspirated and Ja
gelatin, protease inhibitors, and Triton X-114 were from Sigma Chemical the basolateral medium was collected, supplemented with 100 #Ci fresh nua
Co. (Deisenhofen, FRG). 35Smethionine (800 Ci/mmol) was from Amer- 35Smethionine, and saved. After virus adsorption, fresh metabolic label- ry 2
sham Buckler GmbH (Brannschweig, FRG). Reagents for two-dimensional ing medium was added to the apical side and the basolateral medium was 02
gel electrophoresis were obtained from the following vendors: agarose stan- readded for the duration of the infection. 3
dard EEO (Mr, 0.16), Servalyt pH 5-7, and DTT from Serva (Heidelberg,
FRG); Ampholines pH 5-7 and 3.5-10 from LKB Pharmacia (Freiburg, elciseV Isolation from Perforated Cells
FRG); acrylamide and N,N'-methylenabisacrylamide for IEE N,N,N',N'-tet-
rarnethylethylene diamine, ammonium persulfate and SDS from BioRad Perforation of the 100-ram filters was done essentially as described for the
(Munich, FRG); acrylamide and N,N'-methylenebisacrylamide for SDS- 24-ram filters (Bennett et al., 1988) with a few minor changes. The filters
PAGE from British Drug House (Poole, England); NP-40 from Fluka Neu- were excised from the holders and transferred to 14-cm dishes containing
Ulm; urea (enzyme grade) from Bethesda Research Laboratories (Gibeo BRL ice-cold 25 ml KOAc buffer (25 mM Hepes, pH 7.4, 511 mlVl potassium
LTD, Paisley, Scotland); giycine, glycerol, ortho-phosphoric acid, and so- acetate, 2.5 mM MgC12) to wash away cell culture medium. The filter cul-
dium hydroxide from Merck (Darmstadt, FRG). The mouse mAbs directed ture was then placed in a 14-cm dish in a 20°C water bath (in the cold room)
against the cytoplasmic domains of VSV-G protein and PR8 HA were pre- and overlaid with a prewetted nitrocellulose acetate filter (HATF 0.45-#m
pared as described by Kreis (1986) and Hnghson et al. (1988), respectively. pore; Millipore Continental Water Systems, Bedford, MA). Excess mois-
ture was removed by overlaying with a Whatman I filter (Whatman, Kent,
Cell Culture Media England) and smoothing with a bent Pasteur pipette. After binding to the
cells for 90 s the nitrocellulose filter was wetted with 2 rnl KOAc buffer and
Growth medium consisted of EME with Earte's salts (E-MEM) supplemented the filters were carefully separated. After perforation, the filter culture was
with 01 mM Hepes, pH 7.3, 10% (vol/vol) FCS, 100 U/ml penicillin, and transferred to a 10-cm tissue culture dish containing 01 ml GGA buffer (25
100 t~g/ml streptomycin. Infection medium was composed of E-MEM sup- mM Hepes, pH 7.4, 38 mM potassium gluconate, 38 mM potassium gluta-
plemented with 01 mM Hepes, pH 7.3, 0.2% (wt/vol) BSA, 100 U/ml penicil- mate, 38 mM potassium aspartate, 1 mM DTT added fresh, 2 mM EDTA,
lin, and 100 /~g/ml streptomycin. Pulse-labeling medium consisted of and 2.5 mM MgCl:) and an ATP regenerating system consisting of 1 mM
The Journal of Cell Biology, Volume 111, 1990 988
ATE 8 mM creatine phosphate, and 50 t~g/ml creatine kinase. The perfo- supernatant was added. The samples were incubated with rotation at 4"C
rated cells were incubated at 37°C for 1 h after which time the incubation for 3 h (VSV-G protein vesicles) or overnight (WSN HA vesicles ). 1 mg
medium was collected and clarified by centrifugation for 10 min at 1,000 immunoadsorbent (2 mg/rnl in PBS containing 0.1% gelatin) was added and
g~,. The supernatant was overlaid onto a 2-ml 0.25 M sucrose cushion (in the samples were incubated with rotation for an additional 90 rain at 4"C.
10 mM Hepes, pH 7.4, 2 mM EGTA, I mM DTT) in the bottom of an SW40 The cellulose fibers were collected by centrifugation at 4"C and 2,500 rpm
tube and the membranes were pelleted by centrifugation for 3 h at 200,000 in a Microfuge 11 (Beckman Instruments, Inc.). The fibers were washed
*ag and 40C. The supernatant was carefully aspirated with a drawn out three times with 1 ml PBS containing 0.1% gelatin and once with PBS alone.
Pasteur pipette and the wall of the tube was dried with a cotton swab. The After the last wash excess liquid was removed by brief centrifugation in an
membrane pellets were gently resuspended in 300/~1 of 5.1 M sucrose (in Eppendorf microfuge and the bound material was solubilized in 50 ttl gel
I0 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM DTT) with a pipetteman in sample buffer. The third aliquot was diluted with '~3 vol 01 mM Hepes,
preparation for equilibrium density centrifugation. pH 7.4, 2 mM EGTA (to give 0.2-0.25 M sucrose final) and the vesicles
were pelleted by eentrifugation for 3 h at 200,000 ga, and 4"C in a TL-100
Resolution of VSV-G Protein and HA Containing ultracentrifuge (Beckman Instruments, Inc.) using a TLS-55 rotor. The
membrane pellet was resuspended directly in 50/zl sample buffer and served
selciseV on Linear Equilibrium Density Gradients
as the starting material reference.
The membrane pellets were isolated from pulse-labeled VSV or fowl plague
virus-infected cells (three 24-mm MDCK filter cultures used for each vi- SDS-PAGE
rus) after perforation. The pellets were resuspended and mixed in 300 l~t
of 7.1 M sucrose (in 01 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM DTT). SDS-PAGE was performed as described (Bennett et al., 1988). For SDS-
The sample was overlaid with a 0.3-1.5 M linear sucrose gradient and after PAGE immunoisolated samples were solubilized in twofold concentrated
centrifugation for 13.5 h at 125,000 yag in an SW40 rotor 1-ml fractions sample buffer (100 mM "Iris, pH 6.8, 5 mM EDTA, 10% glycerol, 4% SDS,
were collected. Affinity-purified polyclonal antibodies directed against 0.02 % bromo-pbenol blue).
VSV-G protein and fowl plague virus HA were mixed and used to immuno-
precipitate the viral proteins from each gradient fraction as described by Two-dimensional Gel Electrophoresis
Bennett et al. (1988). The centrifuge tube was washed with 1 ml lysis buffer D
o
(20 mM Tris, pH 7.4, 2 mM EDTA, 2 % wt/vol Triton X-100, 0.3 M NaCI, A combination of IEF and SDS-PAGE was used to resolve proteins in two wn
and 01 ttg/ml each of chymostatin, leupeptin, antipaln, and pepstatin) to dimensions essentially as described by Bravo (1984). For two-dimensional loa
d
solubilize pelleted material for immunoprecipitation. gel electrophoresis the samples were solubilized in 9.8 M urea, 4 % (wt/vol) ed
NP-40, 2 % (vol/vol) ampholines pH 7-9 (LKB Instruments, Inc., Gaithers- fro
Purification of selciseV on Discontinuous Equilibrium burg, MD), and 100 mM DTT. The tube gels used for the IEF dimension m h
Density Gradients were 25 cm long and had an internal diameter of 2.5 rnm. IEF gels were ttp
run at 1,200 V for 71 h. The pH gradient after electrophoresis ranged from ://ru
p
All sucrose solutions were prepared in 01 mM Hepes, pH 7.4, 2 mM EGTA, 4.57 to 8.03 and was linear between pH 4.6-7.2. The second dimension re
1 mM DTT. The membrane fractions obtained from up to three 100-mm resolving gels were 15% (wt/vol) acrylamide, 0.075% (wt/vol) N,N'- ss.o
wfaisl ters placed were combiinn ed the bottom in a total of an volume SW60 of tube 300 and 1# 5.1was M overlaids ucrose. with The sample1.9 ml ammeitdhe,y lene-bisacrylamide 0.25 % (wt/vol) N,N'-methylene-bisacrylamidea.n d the stacking gels were 5%A fter (wt/vol) electrophore- acryl- rg/jcb/a
to 1.2 reach M sucrose their equilibriumf ollowed by density 8.1 ml 0.8 by centriM fugation sucrose. The of vetshicel es gradients were for allowed 31 h fsluiosr ography the gels were using fixed Entensify in 45% (Dmueptohnatn,o l aNnEdN 7% Research acetic acid Products, and treated Boston, for rticle-p
at 125,000 yag and 4°C in an SW60 rotor (Beckman Instruments, Inc., Palo MA). df/1
Alto, CA) after which 0.3-ml fractions were collected. The peak fractions 11
containing vesicles were recovered at the 0.8/1.2 M sucrose interface and Quantitation of Two-dimensional Gels /3/9
were pooled. 87
The autoradiographs of two-dimensional gels were digitized using an Os- /10
6
Reagents for Immunoisolation ram CCD video camera and the Image (version 1.16) software program (de- 01
7
veloped by W. Rasband, National Institutes of Health, Bethesda, MD) de- 4
/9
Mouse mAbs directed against the cytoplasmic domains of VSV-G protein signed for use with a Macintosh IIcx computer. The integrated densities of 87
(Kreis, 1986) and influenza PR8 (Hughson et al., 1988) were used in the individual spots were calculated as follows: (N x mean density) - (N x .pd
fwoerrme mainotafi ned concentrated in dense hybridoma culture in culture RPMI supernatantmse. dium containing The hybridomas 10% FCS bagaec kground), density of wherteh e spot, N was and the background number of was pixels, the avemraegaen density density was of the the aver- area f by gu
e
afnodr 2 stored d. The frozen medium in small was harvaelisqtueodt,s for concentrateds ingle use. Cellulose 10-fold by fibers ultrafiitration, were used icamlmceudlaitaet ely the surrporuontdienign recovery the spot. after The immunoisolation integrated densities as follows: were (SID used to- st on 1
aRsi chardson the solid supportan d Luzio, for immunoisolatio1986). n Microgranular (Luzio celeltu lose al., 1976; powder for review was pur- see CsIuDr)e d + for (TID), samples where immunoisolated SID and CID with refer the to specific the integrated and control densities antibody, mea- 4 Jan
u
chehaads,e d 1980). from WhatA man polyclonal and antibody activated directed for protein against coupling the Fc (Hales domain and of Womodo-u se ary 2
0
IgG was raised in sheep by immunizing with purified Fc fragments (pre- 23
pared from mouse IgG digested with immobilized papain as described in
VESICLE PURIFICATION
the Pierce catalog). Sheep antimouse Fc was affinity purified on an Affi-gel
01 matrix (BioRad) with covalently bound mouse IgG and subsequently "Medium" Fraction
coupled to diazocellulose. 1 gan of protein added per mg of diazocellulose
Concentrate by
resulted in 200-300 #g coupled protein. The immunoadsorbent was stable Centrifugation Cytosolic
for several months stored at 4°C as a slurry; 2-rag fibers/ml of PBS contain- (0,25M sucrose cushion) Contaminants
ing 5 mg/ml BSA and 0.02% (wt/vol) sodium azide. Before use the fibers
were washed three times with PBS containing 0.1% gelatin to remove the
Membrane Pellet
BSA and azide.
~
Equilibrium Membrane
lmmunoisolation of selciseV Flotation Contaminants
(Golgi, P.M,etc.)
For each immunoisolation experiment a gradient-purified vesicle fraction
was divided into three equal aliquots. One aliquot was incubated with a con- Vesicle ~ Fraction l ~ l r U
trol antibody and a second aliquot was incubated with the specific antibody,
as follows. Each aliquot was diluted with 0.5 vol PBS containing 0.1% gela- Immunoisolation
tin and a protease inhibitor cocktail (30/~g/rnl each of chymostatin, leupep- Fraction
tin, antipain, and pepstatin) in an Eppendorf tube (Eppendorf/Nethaler, Figure .1 Schematic of vesicle
Hamburg, FRG). To this mixture 10-20 bd concentrated hybridoma culture Specific Vesicles purification.
Wandinger-Ncss et al. igloG-snarT devired-krowteN tropsnarT selciseV 989
tate was collected by centrifugation and dissolved in 50/~1 two-dimensional
gel sample buffer. The detergent phase was washed with 50/~1 cold PBS,
the phases were separated as above, and the aqueous phase was discarded.
The detergent phase was dissolved in 50/zl two-dimensional gel sample
buffer.
EM
Fotrh e morphological characterization of the gradient-purified vesicle frac-
tion, samples were embedded in Epon as follows. A gradient-purified vesi-
cle fraction was fixed in suspension with 2% glutaraldehyde in 120 mM cac-
odylate buffer, pH 7.4, for 30 min at room temperature. The fixed vesicles
werec ollected by centfifugation in an airfuge (Beckman Instruments, Inc.)
for eight rain at 01 psi (60,000 g). The pellet was pestfixed for 30 rain in
a solution of 1.5% osmium tetroxide in cacodylate buffer. After several
washesw ith 50 mM veronal-acetate buffer at pH 7.5, the pellet was stained
for 40 min in the dark using a solution of %1 magnesium uranyl acetate
in 50 mM veronal-acetate buffer, pH 5.2. The sample was dehydrated and
embedded in epon. Ultrathin sections were stained and observed on a
Philips EM301 at 80 .Vk Serial sections were analyzed and confirmed that
the structures shown in Fig. 3 a were vesicles and not cross sections of
tubules.
Immunocytochemistry was performed on ultrathin frozen sections to
identify specific transport vesicleisn the gradient-purified vesicle fraction.
The sucrose gradient fraction containing the vesicles was diluted to 0.25
D
M sucrose and the membranes were collected by centrifugation for 3 h at ow
200,000 yag and 4°C in a TL-100 ultracentrifuge (Beckman Instruments, nlo
a
Inc.) using a TLS-55 rotor. The pellets were fixed with 4% paraformalde- de
hyde in 200 mM Hepes buffer for 5 min followed by fixation with 8% d fro
paraformaldehyde in the same buffer for 1 h. The pellets were infiltrated m
with 2.1 M sucrose in PBS for 20 min and mounted on copper stubs for http
sectioning at -ll0°C using a glass knife and an ultramicrotome (Model ://ru
OMW 4; Reichert, Vienna, Austria) with a cryoattachment. p
re
1984). Immunolabeling The antibody was used performed to label as VSV-G described protein previously containing (Grifliths vesicles et was al., ss.org
a mouse mAb directed against the cytoplasmic domain of VSV-G protein /jcb
(Kreis, 1986). The antibody used to label HA containing vesicles was a /a
mouse mAb specific for the cytoplasmic domain of fowl plague virus HA rticle
(data not shown). For immunolabeling hybridoma culture supernatants -p
d
were diluted 1:1 with 10% FCS. The bound antibodies were vi3ualized with f/1
1
a linker rabbit antimouse antibody followed by protein-A conjugated to 1/3
9 am gold. 25 vesicles of each were analyzed for the diameter measure- /98
ments. 7/1
0
erugiF .2 Gradient-purified vesicles have a characteristic protein 601
7
composition. MDCK cells were metabolically labeled with [35S]- Results 4/9
methionine, infected with VSV, and perforated. A membrane pellet 87
.p
was prepared from the incubation medium and resolved on an equi- Isolation of tropsnarT selciseV from detarofreP slleC df b
librium flotation step gradient. Aliquots from (A) the bottom-most y g
fraction (representing material which did not enter the gradient) MDCK cells grown on permeable filter supports were ue
and from (B) the peak vesicle fraction were analyzed on two- mechanically perforated by overlaying the monolayer with a st on
dimensional gels. nitrocellulose filter, allowing a short drying time and then 14 Ja
gently peeling the filters apart (Bennett et al., 1988, 1989). nu
a
The perforated cells were incubated at 37°C in the presence ry 2
respectively, and TID was the integrated density of the starting material of an ATP regenerating system. After 1 h the incubation 02
3
sample. The recoveries were normalized to the recovery of the respective medium was collected and used to purify the transport vesi-
viral protein, VSV-G protein, or influenza HA and used to calculate the api-
cles for further study. A summary of the purification proce-
cal to basolateral ratios shown in Table II.
dure is shown in Fig. .1
In the first step, transport vesicles and any other mem-
notirT Phase and Extraction X-II4 noitarapeS
branes released during the incubation were concentrated by
centrifugation through a 0.25 M sucrose cushion. This step
Triton X-114 extraction and phase separation were performed by a modifica-
tion of the procedure described by Bordier (1981). The vesicles released also served to remove most of the cytosolic proteins which
from perforated cells were purified on equilibrium step flotation gradients were released from the perforated cells and constituted the
as described above. The peak fractions were pooled and the vesicles were bulk of the protein present in the incubation medium. Next,
pelleted by centrifugation in a TL-100 (Beckman Instruments, Inc.) as de-
the membrane pellet was carefully resuspended in a dense
scribed under Immunoisolation of Vesicles. The vesicle pellet (obtained
from the cells on a single 100-ram filter) was resuspended 5i0n #11% Triton sucrose solution, overlaid with a discontinuous 0.8:1.2 M su-
X-114 (wt/vol) in PBS and transferred to an Eppendorf tube. The sample crose gradient and subjected to equilibrium flotation. It was
was incubated on ice for 51 min, transferred to 37°C for 5 rain to promote our experience that flotation of the transport vesicles to their
phase separation, and then subjected to centrifugation for 1 min in an Ep-
equilibrium density removed residual cytosolicc omponents
pendorf microfuge. The aqueous phase was carefully transferred to a new
and membrane contaminants more effectively than sedimen-
tube with a drawn out Pasteur pipette and the proteins were precipitated
with -20~C acetone and 20 #g BSA added as carrier protein. The precipi- tation. The purification achieved by this step is most clearly
The Journal of Cell Biology, Volume 111, 1990 990
demonstrated by comparing the two-dimensional protein
pattern of the peak vesicle fraction (recovered at the 0.8:1.2
M sucrose interface) with that of the bottom most fraction
(representing material which did not entetrh e gradient). The
samples shown in Fig. 2 were derived from cells metaboli-
cally labeled with [35S]methionine and infected with VSV.
Essentially all of the VSV-G protein, which served as our
transport marker, was recovered in the peak vesicle frac-
tions, while the cytosolic viral nucleocapsidp roteins N and
M remained largely in the bottom fraction (see Fig. 4, lanes
11 and P). In addition,t he overall proteinc omposition of the
vesicle fraction was highly simplified relative to the complex
protein pattern comprising the bottom fraction. Interest-
ingly, many of the proteins in the vesicle fraction appeared
to be sialoglycoproteins as judged by their heterogeneous
charge distribution in the isoelectric focusing dimension (see
also Fig. ,7 A and D). Such proteins constitute only a very
minor fraction of the total cellular protein and are generally
not seen when whole MDCK cell lysates are resolved on
two-dimensional gels (Kondor-Koch et al., 1985).
Analysis of the interface fraction by EM showed it to con- D
o
w
tain primarily vesicles with an average diameter of 100 nm n
lo
(Fig. 3 a). The exocytic vesicles were identified by im- ad
e
d
munogold labeling vesicle fractions isolated from infected fro
cells. Antibodies directed against the cytoplasmic domains m h
of VSV-G protein or influenza HA together with protein-A ttp
gold were used to visualize the vesicles containing these ://rup
re
transport markers on cryosections. A large number of ss.o
cryosectionsp repared from independent samples were ana- rg
lyzed. The gold particles were consistently found clustered /jcb
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on membranes with the expected morphology of carrier vesi- rticle
cles (representative pictures are shown in Fig. ,3 b and c). erugiF .3 Morphology and immunogold labeling of gradient- -p
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No such labeling was observed when the first antibody was section ultrathin an a shows vesicles, transport purified of epon- an f/1
1
omitted (not shown). The average diameter of VSV-G protein from virally isolated fractions fraction. Vesicle vesicle embedded 1/3
containing vesicles determined from such cryosections, was infected cells and purified on flotation equilibrium discontinuous /987
found to be 84 (+11) nm. The average diameter of influenza gradients were Immunocytochem- centrifugation. by concentrated /106
HA vesicles was 78 (+15) nm, not significantly different istry saw used to label cryosections of pellets. The the membrane 017
from that of the VSV-G protein containing vesicles. Thus, in were domains markers transport viral the the cytoplasmic containing against directed membranes antibodies with vesicle beled -alof 4/987
toinolny fromt wo steps, perforated we were MDCK able to cells. isolate This a crufdreac tion vesicle contained frac- conjugated protein-A with visualized and proteins viral .dlogthe b shows G-VSV protein containing vesicles and c to swohs nm 9 .pdf by g
TGN-derived exocytic transport vesicles and had a distinc- vesicles. HA containing influenza ue
tive protein composition. st o
n
1
4
Resolution of Apical and Basolateral by Vesicles using a mixed vesicle fraction. This was prepared by pooling Jan
Equilibrium Flotation ua
the vesicles isolated from cells separately infected with VSV ry 2
Previous work using MDCK cells had shown that newly syn- or with influenza virus. Here we have used fowl plague 02
3
thesized apical and basolateral proteins were delivered influenza virus, because the HA is cleaved during exit from
directly to the appropriate plasma membrane domain after the TGN into the disulphide-linked HA1 and HA2 subunits
exit from the TGN (Matlina nd Simons, 1984; Misek et al., (Matlin and Simons, 1983; Bennett et al., 1988; Wandinger-
1984; Rindler et al., 1985; Pfeiffeert al., 1985; Caplan et Ness and Simons, unpublished observations). This has the
al., 1986). This predicted the existence of two vesicular car- advantage that HA cleavage can be used as a marker to dis-
riers with the necessary information to target proteins to the tinguish the protein remaining in the Golgi (uncleaved) from
respective domain. Therefore, it was of interest to specifical- that present in transport vesicles (cleaved). After perfora-
ly isolate putative apical or basolateral vesicles from the tion, the membrane fractions released from infected cells
crude vesicle fraction for comparison. The spike glyco- were pooled, concentrated, and overlaid with a linear 0.3-
proteins of enveloped RNA viruses were used as transport 5.1 M sucrose gradient. Allowing the vesicles to reach their
markers to tag specific vesicle fractions. Influenza HA served equilibriduemn sity by flotation rather than by sedimentation
as the apical transport marker and VSV-G protein as the allowed us to detect a slight but reproducible difference in
basolateral marker. the densities of apical and basolateral vesicles, marked by the
Previously, we failed to detect any differences in the densi- HA2 and VSV-G proteins, respectively (Fig. 4, A and B).
ties of HA and VSV-G protein containing vesicles (Bennett The peak of HA2 (fraction 5) corresponded to a density of
et al., 1988). However, we have repeated the experiment 1.099 g/ml and that of VSV-G protein (fraction 6) to a density
Wandinger-Ness et al. igloG-snarT devired-krowteN tropsnarT selciseV 199
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erugiF .4 protein and VSV-G HA equilibrium different densities. have containing vesicles The membrane pellets isolated from pulse-labeled d fro
VSV or plague fowl virus-infected cells were resuspended together 0.3-1.5 with M a overlaid and linear sucrose gradient. After centrifuga- m h
tion to equilibrium l-ml fractions were collected and the viral proteins immunoprecipitated from each fraction as well as from the ttp
resuspended pellet. The immunoprecipitates were resolved on a %01 SDS-polyacrylamide gel in the presence of 8 M urea (Bennett et ://ru
p
al., 1988) and the corresponding autoradiogram is shown in A. The lane numbers correspond to the fraction numbers, where one is the re
top of the gradient and P represents the pellet fraction. The lanes marked F and correspond V to delebal-S53 plague fowl virus and VSV ss.o
rg
viral standards, respectively. The viral proteins are indicated. 1AH migrates as is and band diffuse a poorly resolved from protein. N There- /jcb
fore, HA2 is used to the follow cleaved form of HA. The autoradiogram A quantitated was shown in by densitometric scanning. B shows /a
the amounts of HA2 nepo( )selcric protein VSV-G and (filled )selcric in each percentage a expressed fraction as of the total protein recovered rticle
on the gradient. 90% of the total HA was present in the cleaved form. -pd
f/1
1
1
/3
/9
8
7
of 1.113 g/ml. As measured by densitometric scanning, 90 % antibody in solution. Subsequently, cellulose fibers with /10
6
of the total HA recovered on the gradient was of the cleaved covalently bound sheep antimouse Fc antibodies were added 01
7
form and was primarily distributed in fractions 4-7 with very as the solid support. After binding, the fibers were recovered 4/9
8
little HA2 seen at the bottom of the gradient (Fig. 4 B). The by centrifugation and the bound material was solubilized in 7.p
d
small amount of uncleaved HA remained largely in the sample buffer for analysis by gel electrophoresis. Ine ach im- f b
pellet. munoisolation experiment, the vesicle fraction was divided y gu
e
These results provided the first evidence that apical and into three equal samples. One sample was incubated with the st o
n
basolateral membrane proteins might be transported in dis- specific antibody. A second sample was incubated with an 1
4
tinct carrier vesicles. However, since their densities were too Ja
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similar to allow purification on this basis, we took advantage a
of the availability of mAbs directed against the cytoplas- ry 20
2
mic domains of VSV-G protein and influenza HA to selec- IMMUNOISOLATION 3
tively immunoisolate specific vesicle fractions (Kreis, 1986;
Hughson et al., 1988). An mAb directed against the cyto-
plasmic domain of HA derived from influenza strain PR8 has
been well characterized (Hughson et al., 1988). This anti- lanolconoM :bA
Mixed Vesicles ~xHA-COOH ro
body also recognizes the HA of WSN ts61, a temperature-
G-VSV "tail"
sensitive variant with a cytoplasmic domain identical to that
bA gnidniB
~
of PR8 (Ueda and Kilbourne, 1976; Winter et al., 1981; ni Solution
Nakajima et al., 1986). Therefore, these viruses were used
in the following experiments. 0 0
Purification of Transport Vesicles by lmmunoisolation Goat Anti-mouse cF
Cellulose Fibers
Binding to
The strategy we used to immunoisolate specific vesicle frac- ~
Solid Support
tions is shown in Fig. 5 (see also Materials and Methods).
Briefly, a vesicle fraction, purified on a discontinuous flota- erugiF .5 Schematic of vesicle
tion gradient, was first incubated with the specific or control immunoisolation.
The Journal of Cell Biology, Volume 111, 1990 992
L Proteins Table Viral Are Recovered Efficiently
by Immunoisolation
% Recovery (overnight
% Recovery (3 h binding)* binding)
Viral protein Specific Control Specific Control
VSV-G
erugiF .6 G-VSV protein and HA recovered are vesicles containing (n = 5)* 78 + 24 9.7 + 9.6 ND ND
by immunoisolation with antitail antibodies. A gradient-purified PR8 HA
fraction vesicle saw with infected cells pulse-labeled prepared from (n = 2) 13 + 2.2 6.8 + 2.8 40 + 3.0 17 + 5.2
VSV or influenza 8RP HA. containing Vesicles protein VSV-G WSN HA
(lanes 1-3) or HA (lanes 4-6) antibod- with immunoisolated were (n = 3) ND ND 61 + 16 9.3 + 4.2
domains cytoplasmic the against directed ies of viral respective the
* Both densitometric scanning of autoradiographs (Materials and Methods)
proteins. Samples containing G-VSV protein were incubated with and liquid scintillation counting (Bennett et al., 1988) were used to quantitate
antibodies primary the for containing those 4°C and at h 3 HA were the proteins ogne ls. Percent recoveries in specific and control samples are rela-
at incubated 4°C. overnight (lane 1) G-VSV protein recovered on tive to the amount of protein in the starting material sample.
n represents the number of data sets.
the using fibers cellulose G-VSV-itna 2) (lane protein tail antibody.
Background level of G-VSV protein recovered in cellulose fibers
antibody specific the using for 8RP control. a HA as (lane 3) Total
G-VSV used material starting the in present protein for immunoiso- are indeed contained in separate vesicles is that the protein
lation. (lane 4) 8RP HA protein recovered on using fibers cellulose composition of the vesicles should be distinct. Therefore, we
anti-HA the tail (lane antibody. 5) level Background of HA recov- analyzed the protein patterns of the immunoisolated vesicle D
o
ered on using fibers the antibody specific for G-VSV protein as a w
fractions on two-dimensional gels. For this purpose, MDCK n
control, dane 6) Total HA present the material in starting used for cells were metabolically labeled overnight with [35S]methi- load
immunoisolation. ed
onine. The cells were then infected with WSN ts61 or VSV fro
and labeling was continued during infection. The cells were m h
irrelevant antibody as a control for nonspecific binding. A dpifeferrefntoiarl ated, cenatnrdi fugation transport vesicalned immunoisolation. fractions were isolated by ttp://rup
ttehriiradl sample reference. was left untreated and served as the starting ma- (WSN The ts61) typical containing patterns obvtesaiicnleesd are shown for VSV-G in Fig. protein .7 anTd he HA top ress.org
Using this procedure, 78 (+24)% of the total VSV-G pro- panels A( and D) show the protein patterns of the crude vesi- /jcb
tceoivne red and 40 on (+3)% the cellulose of the total fibers PR8 under HA were optimal specifically conditions re- calned fraction E) show used the for proteins immunoisolation. specifically The immunoisolated middle panels to- (B /article-p
d
(Fig. 6 and Table I). The amount of nonspecific binding to gether with the viral proteins. The bottom panels (C and F) f/1
1
the fibers was (cid:127)10% and was similar for both HA and VSV- show the protein patterns resulting from nonspecific binding 1/3
G protein containing vesicles. Comparing several different to the cellulose fibers. The viral proteins are indicated for /987
solid supports for immunoisolation, cellulose fibers were reference in the top panels. VSV-G protein and a soluble /106
found to give good yields with the lowest nonspecific bind- form, sG (Garreis-Wabnitz and Kruppa, 1984; Graeve et 017
4
ing. The lower recovery of PR8 HA as compared to VSV-G al., 1986), are sialylated heterogeneously (Fuller et al., /98
7
protein may be due to differences in antibody affinity. We 1985) and hence are seen as multiple spots (Fig, 7 A). The .pd
htahvee specific varied boatnthi body the temperatinu re order to improvea nd time the of irnecucboavteiorny of with HA cnhoatr ge be attrhiebtuetreodg eneity to differential observed for WsSiNal ylation HA due to (Fig. the 7 D) action can f by gue
containing vesicles. Overnight incubation at 4°C gave the of the viral neuraminidase (see below) and is most likely due st o
n
best results (Table I). Reduced antibody accessibility due to to sulfation (Pinter and Compans, 1975). Its migration as a 14
the relatively small cytoplasmic domain of HA (only 10 doublet was indicative of the factth at WSN ts61 HA was not Jan
u
atmhiisn o reason, acids) vesicles may also were account also isofloatre d the lower from reccoevlelrsy . infected For unH iformly sensitive glycosylatede ven though and both some forms remained were detected endoglycosidase at the cell ary 202
with WSN ts61. In this strain, transport of the HA from the 3
surface (data not shown). The ratio of the two forms varied
ER to the Golgi is temperature sensitive and HA can be
inc ells infected witdhi fferent virus stocks, buhta d no appar-
reversibly accumulated in the ER at nonpermissivet empera-
ent effect on the immunoisolation results. Heterogeneous
tures (Ueda and Sugiura, 1984; Rodriguez-Boulan et al.,
glycosylation ofWSN ts61 HA has been observed previously
1984; Rindler et al., 1985). Upon shifting the cells to per-
(Palese, 1977; Rodriguez-Boulan et al., 1984) and may be
missive temperatures the HA is transported synchronously
attributed to an alteration of the tertiary protein structure by
to the Golgi and its exit from the TGN is blocked by incubat-
the mutation causing the temperature-sensitive phenotype
ing at 20°C (Rodriguez-Boulan et al., 1984; Hughson et al.,
(Nakajima et al., 1986).
1988). The increased antigen concentration in the TGN and
The protein patterns of the crude vesicle fractions ob-
in theb udding transport vesicles may account for the higher
tained from VSV as compared to influenza-infected cells
recoveries (61 + 16%) of HA vesicles obtained with the
were very similar (Fig. ,7 A and D). The most obvious dif-
temperature-sensitive influenza strain (Table I).
ference was the apparent shift in the migration of several gly-
coproteins toward more basic regions of the isoelectric
Protein Composition of Apical and Basolateral
focusing gel when the samples were isolated from influenza-
Vesicle Fractions
infected cells (proteins indicated with arrowheads). This was
Onee xpectation if apical and basolateral membrane proteins most likely caused by the action of the viral neuraminidase
sseN-regnidnaW et al. Network-derived Trans-Golgi Transport Vesicles 993
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Figure .7 nietorP immunoisolatcd of composition lacipa and laretalosab elcisev .snoitcarf A deifirup-tneidarg elcisev noitcarf was detalosi
from VSV (A-C) ro WSN det'.~efni-16st sllec (D-F) and divided otni eerht equal .stouqila The selcisev ni one touqila were detartnecnoc
by noitagufirtnec and served sa eht gnitrats lairetam ecnerefer (A and .)D A second touqila was detabucni with eht ydobitna detcerid tsniaga
eht cimsalpotyc domain of VSV-G .nietorp This antibody was used ot yllacificeps immunoisolate VSV-G nietorp gniniatnoc selcisev )B(
or sa a lortnoc rof cificcpsnon of binding WSN HA gniniatnoc selcisev .)F( A driht touqila was detabucni with eht ydobitna detcerid tsniaga
eht cytoplasmic domain of WSN HA. antibody This was used ot yllacificeps immunoisolate WSN HA gniniatnoc selcisev )E( or sa a
lortnoc rof cificcpsnon of binding VSV-G nietorp gniniatnoc selcisev (C). nI A and arrows small D, etangised eht lariv ,snietorp while
arrowheads etacidni snietorpocylgolais whose ytilibom si deretla ni eht of presence azneulfni .esadinimaruen nI B and E, egral arrows
etacidni snietorp detaicossa yllaitnereferp with VSV-G nietorp arrows) (open ro HA gniniatnoc selcisev dilos( arrows), .ylevitcepser Arrow-
heads etacidni snietorp detaicossa with both VSV-G nietorp and HA gniniatnoc .selcisev The snoitcerid fo IEF and SDS siscrohportcele
sa cra .detacidni An ksiretsa marks eht noitisop fo .nitca The noitisop of delebal-C4L included markers weight molecular ni eht second
dimension era detacidni by )>( on eht tfel hand edis of lla eht .sleg The markers from top ot bottom :era phosphorylasc kD), (200 myosin
kD), (92.5 b BSA 96( ovalbumin kD), 64( anhydrase kD), carbonic 03( kD), and lysozymc 3.41( kD).
The lanruoJ fo lleC ,ygoloiB Volume ,III 0991 994
Table .11 Quantitation of Vesicle Proteins Identified on
Two-dimensional Gels
Protein Apical/Basolateral *oitar loM wt~: Localization§
A3 3.41 108,000 peripheral
A4 6.0 92,000 peripheral
A5 8.01 91,000 peripheral
A 41 3.51 65,000 peripheral
A 61 1.01 69,000 peripheral
A23 38.0 46,000 membrane
A26 20.0 29,000 membrane
B4 0.48 54,000 peripheral
5B 0.11 52,000 peripheral
8B 0.42 35,000 membrane
B9 0.42 24,000 membrane
BI2 0.14 23,000 membrane
C3 9.1 68,000 peripheral
C4 3.1 66,000 membrane
C8 2.2 54,000 peripheral
C9 2.1 39,000 membrane
CI0 2.6 27,000 membrane D
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C 11 3.1 24,000 membrane a
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C13 2.4 20,000 membrane d
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* cirtemotisneD gninnacs saw to used etatitnauq owt data stes htiw deniatbo h
hcae virus dna seirevocer were detaluclac sa debircsed ni slairetaM dna ttp
.sdohteM ehT lacipa to laretalosab oitar saw detaluclac :sa yrevoceR( evitaler ://ru
to )NSW + yrevoceR( evitaler ot .)VSV pre
:~§ deegasreavBA no eulav notirT denimreted 411-X esahp from .gninoititrap eerht etarapes .sleg ss.org
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clearly documented for VSV-G protein in cells coinfected rticle
with influenza virus (Fuller et al., 1985). -p
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Comparing the immunoisolated samples (Fig. 7, B and E) f/11
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with the starting fractions, a subset of proteins which are /3/9
erugiF .8 Membrane and peripheral vesicle proteins identified by clearly coisolated with the viral proteins can be seen. Pro- 87
/1
Triton X-114 phase separation. Two 100-ram filter cultures of teins that were also not detected to any significant degree in 06
0
MDCK cells were metabolically labeled and infected with VSV. the control samples (Fig. 7, C and F) and were, therefore, 17
4
The cells were perforated and a gradient-purified vesicle fraction considered to be specifically isolated are labeled (arrows and /98
wparse pared. One-half of the vesicle fraction was subjected to Tri- arrowheads). As expected, residual cytosolic proteins, e.g., 7.pd
tresulting on X-114 detergent extraction )A( and and phase aqueous separation. (B) phases were The proteins resolved in the on actin (marked by an asterisk) and the viral nucleocapsid pro- f by g
teins, still present in trace amounts in the vesicle fraction ue
tbwyo -dimensional small arrows. Arrowheads gels. The viral proteins are designate proteinpsr esent labeled and in indicated both api- were not specifically isolated. st on 1
cal and basolateral vesicles, solid arrows mark apical specific pro- It was of primary interest to compare the protein patterns 4 Ja
teins, and open arrows indicate basolateral specific proteins. Note: of HA and VSV-G protein containing vesicles (Fig. 7, B and nu
a
several proteins shown in Figs. 7 and 9 were only seen on a longer E). After a careful comparison of several data sets, it became ry 2
exposure, including: A15 (aqueous), A17 (aqueous), A24 (in both apparent that while there were many similarities there were 02
3
phases), A25 (detergent), BI3 (detergent), and C7 (aqueous). An also differences in the two patterns. A number of proteins
asterisk marks the position of actin and the directions of IEF and were present in both fractions (Fig. 7, B and E, arrowheads).
SDSe lectrophoresis are as indicated. The molecular weight mark-
Several proteins were selectively recovered with HA con-
ers designated by (>) are as in Fig. .7
taining vesicles, regardless of which influenza strain was
used, PR8 or WSN ts61 (Fig. 7 E, solid arrows). Another
group of proteins was specifically associated with the VSV-G
vesicle fraction (Fig. 7 B, open arrows). Interestingly, some
on sialoglycoproteins. The viral neuraminidase is synthe-
of the proteins characterisitic of apical vesicles (Fig. 7 E)
sized and transported together with HA to the TGN where
were less prevalent in the gradient-purified vesicle fractions
it can catalyze the selective removal of sialic acid residues.
isolated from VSV-infected cells (Fig. 7 A). One possible ex-
Since such sialoglycoproteins are usually seen as multiple
planation for this is that fewer apical vesicles are formed in
spots representing different degrees of sialylation the se-
the absence of cargo molecules.
quential removal of sialic acid residues by neuraminidase
results in a reduction of the net negative charge on the pro-
Further Characterization of Vesicle Proteins
teins. Hence their migration in isoelectric focusing gels is al-
tered and fewer spots are observed. This effect has been Additional information about the proteins present in the vesi-
sseN-regnidnaW te .la igloG-snarT devired-krowteN tropsnarT selciseV 599
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Figure .9 Schematic of and apical /9
8
basolateral specific vesicle pro- 7/1
0
A teins. swohs prefer- proteins the 60
1
entially associated with apical 74
vesicles as shaded forms with /98
7
numbers A preceded (apical). by .pd
B tially associated with basolateral shows the proteins preferen- f by gu
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vesicles as shaded forms with st o
n
numbers preceded by B (baso- 1
4
lateral). Filled forms represent Ja
n
api- both with associated proteins u
a
cal and basolateral vesicles and ry 2
0
are designated by numbers pre- 2
3
faced with C (common).
cle fraction was obtained from Triton X-114 phase partition- The recovery of all the proteins after phase separation was
ing. This method has been used extensively to distinguish close to 100% and only the samples representing the deter-
membrane and peripheral proteins (Bordier, 1981). Mem- gent (Fig. 8 A) and aqueous (Fig. 8 B) phases are shown.
brane proteins, with some exceptions, partition into the de- In general, the proteins partitioned into one of the two
tergent phase, whereas peripheral proteins are typically re- phases. One notable exception was actin which was dis-
covered in the aqueous phase. For this experiment, cells tributed equally in both phases (marked by an asterisk in Fig.
were metabolically labeled with [35S]methionine and in- 8, A and B). Since actin is a soluble cytosolic protein, its
fectedw ith VSV. A crude vesicle fraction was prepared and presence in the detergent phase may be best explained by
divided into two equal parts. One sample was left untreated some association with membrane proteins. The fact that ac-
as a starting material reference. The second sample was sub- tin was not significantly recovered in the immunoisolated
jected to Triton X-114 phase partitioning and separated into samples (Fig. ,7 B and E) suggests that it is not associated
an aqueous and a detergent phase. All three samples were with the transport vesicle membranes.
analyzed by two-dimensional gel electrophoresis. The partitioning of proteins into the aqueous or detergent
The Journal of Cell Biology, emuloV lll, 0991 699