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(Received for publication, June 22, 1995; and in revised form, August 31, 1995) From the
Furin is a membrane-associated endoprotease that catalyzes
cleavage of precursor proteins at Arg-X-Lys/Arg-Arg sites.
Although, at steady state, furin is predominantly found in the trans-Golgi network (TGN), it also cycles between the TGN and
the cell surface. Recently, the cytoplasmic tail of furin has been
shown to be sufficient for its localization to the TGN. Within the
cytoplasmic domain, there are Ser residues, which we now show are sites
for phosphorylation by casein kinase II in vitro, and a
Tyr-containing sequence, both of which have been shown to be important
for other TGN proteins to localize to this compartment. In the present
study, we show by site-directed mutagenesis that these residues are
important for TGN localization and recycling of furin. Mutation of the
Ser residues abrogated the TGN localization. By contrast, mutation of
the Tyr residue did not affect the TGN localization but impaired the
internalization from the plasma membrane. These observations suggest
that distinct cytoplasmic determinants are responsible for retention in
the TGN and retrieval from the cell surface of furin.
A novel family of mammalian processing endoproteases homologous
to the yeast Kex2 protease has been shown to play a pivotal role in the
production of a wide variety of biologically active peptides and
proteins. These include furin, PC1/3, PC2, PC4, PACE4, and PC5/6 (for
review, see Seidah et al.(1991), Steiner et al. (1992), and Halban and Irminger(1994)). Furin is a
membrane-associated protease that catalyzes cleavage of a wide variety
of precursor proteins at sites marked mainly by the
Arg-X-Lys/Arg-Arg sequence (Bresnahan et al., 1990;
Hosaka et al., 1991; Misumi et al., 1991). At steady
state, furin is predominantly found in the trans-Golgi network
(TGN), ( The TGN is a
compartment that appears as a tubuloreticular network extending from
the trans-Golgi cisterna and plays a pivotal role in sorting
of proteins within the exocytic pathway (Mellman and Simons, 1992;
Ladinsky et al., 1994). Newly synthesized proteins delivered
through the Golgi stacks to the TGN are packaged into different
transport vesicles, which are directed to the plasma membrane,
lysosomes, regulated secretory granules, and so on (Mellman and Simons,
1992; Bauerfeind and Huttner, 1993). Signals required for
localization of membrane proteins to the TGN have been extensively
investigated for TGN38 and cation-dependent and cation-independent
mannose 6-phosphate receptors (MPRs). In all cases, the cytoplasmic
domain is responsible for the TGN localization (Kornfeld and Mellman,
1989; Kornfeld, 1992; Luzio and Banting, 1993; Stanley and Howell,
1993; Conibear and Pearse, 1994; Mauxion et al., 1995) and is
capable of interacting with cytosolic transport proteins (Glickman et al., 1989; Le Borgne et al., 1993; Jones et
al., 1993). Like furin, TGN38 is predominantly localized to the
TGN and cycles between this compartment and the cell surface (Luzio et al., 1990; Ladinsky and Howell, 1992; Reaves et
al., 1993). By mutagenesis, a Tyr-containing sequence, YQRL, in
TGN38 has been shown to function as the TGN localization signal (Bos et al., 1993; Humphrey et al., 1993; Wong and Hong,
1993). MPRs are mainly localized to the TGN and endosomes and recycle
between these compartments and the plasma membrane (Kornfeld and
Mellman, 1989; Kornfeld, 1992). Signals within the cytoplasmic tail
involved in targeting and recycling of MPRs appear to be somewhat
different from those of TGN38. A Tyr-containing sequence is capable of
interacting with the coat protein complex in the plasma membrane
(HA-II/AP-2 clathrin adaptor) but not that in the TGN (HA-I/AP-1
clathrin adaptor) (Glickman et al., 1989) and is responsible
for internalization from the cell surface rather than retention in the
TGN (Johnson et al., 1990; Canfield et al., 1991;
Conibear and Pearse, 1994; Mauxion et al., 1995). On the other
hand, a Ser residue in the MPR cytoplasmic tail, which can be
phosphorylated by casein kinase II (CK-II), is involved in interaction
with the HA-I/AP-1 adaptor complex and is closely associated with exit
from the TGN (Méresse et al., 1990; Le
Borgne et al., 1993; Méresse and
Hoflack, 1993; Körner et al., 1994). In
the case of furin, there is a Ser-containing sequence, which fits the
consensus sequence for phosphorylation by CK-II (Kennelly and Krebs,
1991), and a Tyr-containing sequence, YKGL, which is reminiscent of
those of TGN38 and MPRs, within the cytoplasmic domain. In the present
study, we therefore examined the roles of the Ser and Tyr residues in
the localization of furin to the TGN and its retrieval from the cell
surface.
As described in the Introduction, within the furin
cytoplasmic domain there is a unique Tyr residue (Tyr-758) and two Ser
residues (Ser-772 and Ser-774), which lie within potential CK-II
phosphorylation sites (Fig. 1). In order to examine whether the
Tyr and Ser residues are important for localization and recycling of
furin, these residues were changed into Ala residues (Fig. 1).
At first, we examined whether the Ser residues were indeed
phosphorylated by CK-II. The cytoplasmic tail sequence with or without
the mutation was fused with GST. The GST-furin tail fusion protein was
then expressed in E. coli, purified using
glutathione-Sepharose, and incubated with recombinant CK-II in the
presence of [
Figure 1:
Schematic representation of the
structures of furin tail fusion protein and its mutants. Substituted
residues in the furin tail of the mutants are indicated. WT,
wild type; Fur, furin; TM, transmembrane domain; CT, cytoplasmic tail.
Figure 2:
Phosphorylation by CK-II. GST(-) or
GST-fusion protein with wild type (WT) furin tail or with the
tail having SA or YA mutation was incubated with CK-II and
[
To examine the effects of these mutations on
steady state localization of furin, the furin sequence containing the
transmembrane and cytoplasmic domains with or without the mutations was
fused with the extracellular domain of CD4. The expression vector was
stably transfected into human hepatoma HepG2 cells. Indirect
immunofluorescence microscopy with anti-CD4 monoclonal antibody
revealed that strong perinuclear staining characteristic of the Golgi
was observed for the CD4 fusion protein with the wild type furin
sequence (Fig. 3, WT) when the cells were permeabilized
after fixation (P). When the permeabilization step was omitted (NP), no staining was observed. Essentially identical results
were obtained using another chimeric construct in which the cytoplasmic
domain of furin was attached to the ecto- and transmembrane domains of
CD4 (data not shown). These observations are in agreement with those of
others (Bosshart et al., 1994; Chapman and Munro, 1994) and
indicate that the cytoplasmic domain is sufficient to confer the TGN
localization. In a very small fraction of cells with most intense
Golgi-like staining, some punctate staining was also observed
throughout the cytoplasm under permeabilized conditions, and very weak
cell surface staining was observed under non-permeabilized conditions
(data not shown). These observations were similar to those reported by
others (Bosshart et al., 1994) and suggest a saturable
mechanism for the TGN localization of furin as proposed in the case of
TGN38 (Wong and Hong, 1993; Reaves and Banting, 1994). Steady state
localization of the CD4-furin tail fusion protein with the YA mutation
appeared not to be significantly different from that of wild type (Fig. 3, YA). By contrast, in HepG2 cells stably
transfected with the construct having the SA mutation (Fig. 3, SA), perinuclear Golgi-like staining was no longer observed
under permeabilized conditions. Instead, punctate structures throughout
the cytoplasm were predominant. These cytoplasmic vesicles probably
correspond to endosomes as reported by others (Bosshart et
al., 1994; Chapman and Munro, 1994). Under non-permeabilized
conditions, moderate cell surface staining was observed. In cells
transfected with the construct having mutations at both Tyr and Ser
residues (Fig. 3, YA/SA), more intense cell surface
staining as well as punctate cytoplasmic staining was observed. We also
transiently transfected the fusion constructs with or without the
mutations into monkey kidney COS-7 cells and observed essentially the
same staining pattern for each construct (Fig. 4).
Figure 3:
Steady state localization of CD4-furin
tail chimeras in stably transfected HepG2 cells. HepG2 cells stably
expressing CD4-fusion protein with the wild type (WT) furin
tail or with the tail having YA, SA, or YA/SA mutation were fixed and
processed for immunofluorescence analysis using anti-CD4 monoclonal
antibody as described under ``Experimental Procedures.'' Upper panels, cells permeabilized after fixation (P); lower panels, cells without the permeabilization step (NP).
Figure 4:
Steady state localization of CD4-furin
tail chimeras in transiently transfected COS-7 cells. COS-7 cells
transiently expressing CD4-fusion protein with the wild type or mutant
furin tail were stained using anti-CD4 monoclonal antibody. Details and
abbreviations are similar to those in the legend to Fig. 3.
To
investigate the effects of the above mutations on recycling of the
fusion protein from the cell surface, we then performed antibody uptake
experiments. COS-7 cells transiently transfected with the fusion
construct were incubated with anti-CD4 monoclonal antibody in culture,
fixed, and processed for immunofluorescence microscopy. As shown in Fig. 5, cells transfected with the CD4-wild type furin tail
construct internalized the antibody and localized it predominantly to a
perinuclear Golgi region. This result was in good agreement with that
reported by others (Molloy et al., 1994; Chapman and Munro,
1994). In cells expressing the fusion protein with the YA mutation, the
antibody uptake was inefficient. Non-permeabilized cells showed intense
cell surface staining. Furthermore, many endosome-like vesicles were
stained, although a perinuclear region was also significantly stained.
These results suggest that the YA mutation causes retardation in
endocytosis of the fusion protein from the plasma membrane. By
contrast, in cells expressing the fusion protein with the SA mutation,
cell surface staining was barely detectable. However, no Golgi-like
staining was observed, and endosome-like staining was prominent. These
observations suggest that although the SA mutant is indeed endocytosed
from the plasma membrane to endosomes, it is unable to move from
endosomes to the TGN. The staining pattern observed in cells expressing
the chimera with both YA and SA mutations appeared to be most different
from that in wild type-expressing cells. Intense cell surface staining
and no Golgi-like staining were observed.
Figure 5:
Recycling of CD4-furin tail chimeras in
transiently transfected COS-7 cells. COS-7 cells transiently expressing
CD4-fusion protein with the wild type (WT) or mutant furin
tail were incubated with anti-CD4 monoclonal antibody in culture,
fixed, and processed for immunofluorescence analysis as described under
``Experimental Procedures.''. Upper panels, cells
permeabilized after fixation (P); lower panels, cells
without the permeabilization step (NP).
Furin was first found to be predominantly localized to the
TGN, but more recent studies have shown that it also cycles between the
TGN and the cell surface (Molloy et al., 1994) and that its
cytoplasmic tail is necessary and sufficient to confer the correct
localization and recycling (Molloy et al., 1994; Chapman and
Munro, 1994; Bosshart et al., 1994). We noted that the
cytoplasmic domain of furin contains (i) a unique Tyr residue within a
sequence reminiscent of previously described Tyr-containing TGN
localization motifs and (ii) two Ser residues that lie within consensus
CK-II sites. In the present study, we examined if these residues were
important for TGN localization and recycling by expressing chimeras of
the CD4 ectodomain and the transmembrane and cytoplasmic domains of
furin. When the Tyr was mutated to Ala, retention of the fusion
protein in the TGN appeared not to be impaired. However, its retrieval
from the plasma membrane to the TGN was significantly retarded (Fig. 5). A similar phenomenon has been reported in the case of
MPRs, which are also present in the TGN and cycle between this
compartment and the cell surface (Conibear and Pearse, 1994; Mauxion et al., 1995). Previous studies have established that there
are internalization signals containing unique Tyr residues within the
cytoplasmic domains of constitutively recycling receptors (for review,
see Trowbridge (1991), Luzio and Banting(1993), and Sandoval and
Bakke(1994)). When receptors such as low density lipoprotein and
transferrin receptors bind their ligands at the plasma membrane, they
undergo rapid internalization and are delivered to endosomes. Here they
release their bound ligands and recycle back to the cell surface. Thus,
the Tyr-containing sequence is a signal for delivery of these receptors
from the cell surface to endosomes. The data we presented here show
that this is also true for furin. We confirmed that the Ser residues
can be phosphorylated by CK-II in vitro. The data presented
here indicate that the Ser residues are important for the TGN
localization of furin, although it is unknown whether these residues
are phosphorylated in vivo. When the Ser residues were mutated
to Ala, the CD4-furin chimera was no longer retained in the TGN;
instead it was predominantly found in endosome-like structures and at
the plasma membrane. An antibody uptake experiment showed that the SA
mutant chimera was indeed internalized from the cell surface but did
not reach to the TGN. The delivery of the chimera from the cell surface
appeared to be blocked in an endosomal compartment. That furin passes
through endosomes on its way from the cell surface to the TGN has been
demonstrated by the recent data of Chapman and Munro(1994). They have
shown that internalization of a furin chimera is blocked at endosomes
when cells expressing the chimera are treated with chloroquine, which
neutralizes acidic organelles. In this concern, the data of Bosshart et al.(1994) are also noteworthy. They have provided evidence
that a fraction of furin molecules is delivered to a lysosomal
compartment for degradation, although it is unclear whether routing of
furin to lysosomes occurs directly from an internal site or involves a
round of transport through the cell surface. Taken together, we
postulate either or both of the following possibilities for the role of
the Ser residues. One is that the Ser residues are essential for
retention of furin molecules in the TGN. This is supported by the data
that the SA mutant is localized to endosomes and the plasma membrane at
steady state. The other possibility is that the Ser residues are
involved in retrieval from endosomes to the TGN of furin molecules,
which have gone out of the TGN to endosomes or are internalized from
the plasma membrane to endosomes. The observations for the YA/SA
mutant support the above mentioned roles of the Tyr and Ser residues.
Both retention in the TGN and internalization from the cell surface of
the YA/SA chimera were severely impaired. Fig. 6illustrates the
models for trafficking of furin and its mutants on the basis of the
observations presented here and the data of others (Chapman and Munro,
1994; Bosshart et al., 1994). Basically, the majority of furin
molecules are retained in the TGN. A saturable mechanism may underlie
the retention as proposed in the case of TGN38 (Hong and Wong, 1993;
Reaves and Banting, 1994). A fraction of the molecules released from
the TGN retention may be delivered to the plasma membrane and cycle
back to the TGN via endosomes and/or may be directly delivered to
endosomes. Then, the molecules in endosomes may return to the TGN. Our
data indicate that, to confer correct TGN localization, the cytoplasmic
tail of furin contains at least two signals: the Tyr residue that is
involved in internalization from the cell surface to endosomes and the
Ser residues that can be phosphorylated by CK-II in vitro and
are involved in retention in and retrieval from endosomes to the TGN.
However, whether phosphorylation of these residues regulates
localization of furin in vivo is yet to be determined.
Figure 6:
Models for intracellular trafficking of
furin and its mutants. Heavier arrows indicate heavier
intracellular traffic. WT, wild
type.
In
MPRs, HA-II/AP-2 plasma membrane adaptor has been shown to interact
with a Tyr-containing sequence (Glickman et al., 1989), and
HA-I/AP-1 Golgi adaptor has been suggested to recognize another region
of the cytoplasmic tail, probably the Ser residues of the consensus
CK-II sites (Glickman et al., 1989; Le Borgne et al.,
1993). Furthermore, Méresse et al.(1990)
have shown that the Ser residues are phosphorylated by a CK-II-like
activity associated with the HA-I/AP-1 adaptor. These data are
compatible with the notion that the Tyr and Ser residues are involved
in retrieval from the plasma membrane and retention in the TGN,
respectively, of MPRs. Taken together with the data presented here, we
think that similar mechanisms underlie localization to the TGN and
recycling of furin. By contrast, in the case of another TGN-resident
protein, TGN38, the Tyr-containing motif appears to be responsible for
both retention in the TGN and retrieval from the cell surface (Bos et al., 1993). However, there is a report showing that a Ser
residue, which is positioned two residues upstream of the Tyr, is also
important for the TGN retention (Hong and Wong, 1993). Furthermore,
Ponnambalam et al.(1994) have shown that not only the
cytoplasmic tail but also the transmembrane domain of TGN38 is
implicated in its correct localization. Thus, the localization
mechanisms of TGN38 may be somewhat different from those of furin and
MPRs. Recently, a protein complex including an unidentified protein of
62 kDa and Rab6 has been reported to be capable of binding the TGN38
cytoplasmic tail (Jones et al., 1993). However, it has not
been examined if the cytoplasmic tail can interact with the clathrin
adaptor complexes. Further analyses on interaction between the
cytoplasmic tail of the TGN membrane proteins and cytosolic factors
will be required to understand the mechanisms underlying retention in
the TGN and recycling of these proteins.
Volume 270,
Number 47,
Issue of November 24, 1995 pp. 28397-28401
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)where it undergoes endoproteolysis (Misumi et
al., 1991; Molloy et al., 1994). In addition, lines of
evidence have suggested that a fraction of furin is present on the cell
surface (Klimpel et al., 1992; Tsuneoka et al.,
1993). Recent observations indicate that furin cycles between the cell
surface and the TGN (Molloy et al., 1994). Furthermore,
analyses using deletion mutants and chimeric proteins have indicated
that targeting to the TGN and retrieval from the cell surface of furin
require its cytoplasmic tail (Molloy et al., 1994; Chapman and
Munro, 1994; Bosshart et al., 1994).
DNA Constructions
A BamHI site was
introduced at the start of the cytoplasmic domain and a XhoI
site after the stop codon of mouse furin cDNA (Hatsuzawa et
al., 1990) by polymerase chain reaction (PCR)-based techniques.
This BamHI-XhoI fragment was cloned into a
glutathione S-transferase (GST) fusion vector, pGEX-4T-2
(Pharmacia Biotech Inc., Uppsala, Sweden). A HindIII-ApaLI fragment covering the extracellular
domain of human CD4 and an ApaLI-XhoI fragment
covering the transmembrane and cytoplasmic domains of mouse furin,
which were both made by PCR-based techniques, were cloned together
between the HindIII and XhoI sites of a mammalian
expression vector, pcDNA3 (Invitrogen, San Diego, CA). YA and SA
mutants of the furin cytoplasmic tail were constructed by PCR of the
cDNA fragment in pGEX-4T-2 using 5` primers, which modified the
sequence. The sequences of all the constructs were verified by DNA
sequencing.Phosphorylation by CK-II
Escherichia coli JM109 cells carrying the vector for the GST-furin tail fusion
protein were cultured to an A of 0.6-0.8
in 5 ml of L broth and then treated with 1 mM isopropyl
-D-thiogalactoside for 3 h. The cells were sedimented,
resuspended in 2.5 ml of ice-cold phosphate-buffered saline, and lysed
using a sonicator. The cell lysate was centrifuged to remove insoluble
material. Approximately 20 µl of a 50% slurry of
glutathione-Sepharose 4B (Pharmacia Biotech Inc.) was added to 200
µl of the supernatant and mixed gently for 1-2 h at room
temperature. The Sepharose beads were sedimented, washed five times
with CK-II buffer (20 mM MES-KOH, pH 6.9, 130 mM KCl,
10 mM MgCl
, 4.8 mM dithiothreitol), and
resuspended in 50 µl of CK-II buffer. The suspension was then
incubated with 10 µCi of [-
P]ATP (3000
Ci/mmol; Amersham, Buckinghamshire, UK) and 0.1 milliunit of
recombinant CK-II (Boehringer Mannheim GmbH, Mannheim, Germany) at 37
°C for 1 h. The beads were then washed five times with CK-II buffer
and resuspended in SDS-gel sample buffer containing 2-mercaptoethanol.
The suspension was boiled for 5 min and centrifuged. The supernatant
was electrophoresed on a 12.5% SDS-polyacrylamide gel and subjected to
autoradiography.
DNA Transfection and Indirect Immunofluorescence
Microscopy
The expression vector for the chimera of CD4 and
furin was transfected into HepG2 or COS-7 cells by using a CellPhect
transfection kit (Pharmacia Biotech Inc.). For HepG2 cells, stable
transfectants were then selected in the presence of 0.4 mg/ml G418
(Sigma). Pools of G418-resistant HepG2 cells and COS-7 cells 48 h after
transfection were trypsinized, plated onto wells of eight-well Lab-Tek
chamber slides (Nunc, Roskilde, Denmark), and cultured for 12 h. The
cells were fixed, permeabilized, and blocked as described previously
(Torii et al., 1995). In some cases, the permeabilization step
was omitted. The cells were then incubated sequentially with monoclonal
mouse anti-CD4 antibody (Leu-3a; Becton Dickinson Immunocytometry
Systems, San Jose, CA) and fluorescein isothiocyanate-labeled donkey
anti-mouse IgG (Chemicon, Temecula, CA). In antibody uptake
experiments, cells were exposed to Leu-3a antibody (5 µg/ml) in
culture for 1 h. The cells were then fixed, permeabilized, blocked, and
treated with secondary antibody. In some cases, the permeabilization
step was omitted. The stained cells were observed with a laser-scanning
confocal microscope (TCS4D, Leica Lasertechnik, GmbH, Heidelberg,
Germany).
-
P]ATP. As shown in Fig. 2, the fusion proteins with the wild type furin tail and
with the tail having the mutation of Tyr to Ala (YA mutant) were
phosphorylated by CK-II. Although the identity of a minor band that
migrated a little faster than the GST-fusion protein in the SDS gel is
not known, it is most likely to be a degradation product of the
GST-furin tail fusion protein. By contrast, the fusion protein with
mutation of the two Ser residues to Ala (SA mutant) was not
phosphorylated. These results indicate that either or both of the two
Ser residues (Ser-772 and Ser-774) can be phosphorylated by CK-II in vitro.
-
P]ATP, electrophoresed on
SDS-polyacrylamide gel, and subjected to autoradiography as described
under ``Experimental Procedures.'' The position of GST-furin
tail fusion protein is indicated by an arrow.
)
We thank Dr. Hiromitsu Nakauchi for providing the CD4
cDNA, Drs. George Banting and Yukio Ikehara for critical comments on
the manuscript, and Dr. Kaichiro Yanagisawa for encouragement.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y. Xiang, S. S. Molloy, L. Thomas, and G. Thomas The PC6B Cytoplasmic Domain Contains Two Acidic Clusters That Direct Sorting to Distinct trans-Golgi Network/Endosomal Compartments Mol. Biol. Cell, April 1, 2000; 11(4): 1257 - 1273. [Abstract] [Full Text]
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 C. E. Gustafson, T. Katsura, M. McKee, R. Bouley, J. E. Casanova, and D. Brown Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive trans-Golgi-associated compartment Am J Physiol Renal Physiol, February 1, 2000; 278(2): F317 - F326. [Abstract] [Full Text] [PDF]
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R. T. Watson and J. E. Pessin Functional Cooperation of Two Independent Targeting Domains in Syntaxin 6 Is Required for Its Efficient Localization in the trans-Golgi Network of 3T3L1 Adipocytes J. Biol. Chem., January 14, 2000; 275(2): 1261 - 1268. [Abstract] [Full Text] [PDF]
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M. Teuchert, S. Berghofer, H.-D. Klenk, and W. Garten Recycling of Furin from the Plasma Membrane. FUNCTIONAL IMPORTANCE OF THE CYTOPLASMIC TAIL SORTING SIGNALS AND INTERACTION WITH THE AP-2 ADAPTOR MEDIUM CHAIN SUBUNIT J. Biol. Chem., December 17, 1999; 274(51): 36781 - 36789. [Abstract] [Full Text] [PDF]
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B. D. Caldwell, D. N. Darlington, P. Penzes, R. C. Johnson, B. A. Eipper, and R. E. Mains The Novel Kinase Peptidylglycine alpha -Amidating Monooxygenase Cytosolic Interactor Protein 2 Interacts with the Cytosolic Routing Determinants of the Peptide Processing Enzyme Peptidylglycine alpha -Amidating Monooxygenase J. Biol. Chem., December 3, 1999; 274(49): 34646 - 34656. [Abstract] [Full Text] [PDF]
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M. E. Milla, M. A. Leesnitzer, M. L. Moss, W. C. Clay, H. L. Carter, A. B. Miller, J.-L. Su, M. H. Lambert, D. H. Willard, D. M. Sheeley, et al. Specific Sequence Elements Are Required for the Expression of Functional Tumor Necrosis Factor-alpha -converting Enzyme (TACE) J. Biol. Chem., October 22, 1999; 274(43): 30563 - 30570. [Abstract] [Full Text] [PDF]
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A. Bruzzaniti, R. Marx, and R. E. Mains Activation and Routing of Membrane-tethered Prohormone Convertases 1 and 2 J. Biol. Chem., August 27, 1999; 274(35): 24703 - 24713. [Abstract] [Full Text] [PDF]
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Q. Liu, J. H. Grubb, S. S. Huang, W. S. Sly, and J. S. Huang The Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Is a Substrate of Type V Transforming Growth Factor-beta Receptor J. Biol. Chem., July 9, 1999; 274(28): 20002 - 20010. [Abstract] [Full Text] [PDF]
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 T. Simmen, M. Nobile, J. S. Bonifacino, and W. Hunziker Basolateral Sorting of Furin in MDCK Cells Requires a Phenylalanine-Isoleucine Motif Together with an Acidic Amino Acid Cluster Mol. Cell. Biol., April 1, 1999; 19(4): 3136 - 3144. [Abstract] [Full Text] [PDF]
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S. F. Nothwehr, P. Bruinsma, and L. A. Strawn Distinct Domains within Vps35p Mediate the Retrieval of Two Different Cargo Proteins from the Yeast Prevacuolar/Endosomal Compartment Mol. Biol. Cell, April 1, 1999; 10(4): 875 - 890. [Abstract] [Full Text]
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I. Stefaner, A. Praetor, and W. Hunziker Nonvectorial Surface Transport, Endocytosis via a Di-leucine-based Motif, and Bidirectional Transcytosis of Chimera Encoding the Cytosolic Tail of Rat FcRn Expressed in Madin-Darby Canine Kidney Cells J. Biol. Chem., March 26, 1999; 274(13): 8998 - 9005. [Abstract] [Full Text] [PDF]
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M. Teuchert, W. Schafer, S. Berghofer, B. Hoflack, H.-D. Klenk, and W. Garten Sorting of Furin at the Trans-Golgi Network. INTERACTION OF THE CYTOPLASMIC TAIL SORTING SIGNALS WITH AP-1 GOLGI-SPECIFIC ASSEMBLY PROTEINS J. Biol. Chem., March 19, 1999; 274(12): 8199 - 8207. [Abstract] [Full Text] [PDF]
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