(Received for publication, September 16, 1996, and in revised form, November 7, 1996)
From the UBC6 is a C-terminal membrane-anchored (type IV)
protein, native to Saccharomyces cerevisiae, where it is
found in the endoplasmic reticulum. When expressed in mammalian cells,
this novel ubiquitin-conjugating enzyme also localizes to the
endoplasmic reticulum. UBC6 lacks a lumenal domain and contains no
known endoplasmic reticulum retention signals. Analysis of chimeric
proteins in which the cytosolic domain of UBC is linked to a
heterologous transmembrane domain, or in which the UBC6 transmembrane
domain is appended to an unrelated soluble protein, led to the
determination that the transmembrane domain of UBC6 plays a dominant
role in its compartmental localization. The basis for the transmembrane
domain-mediated subcellular targeting of UBC6 was evaluated by
lengthening the wild type UBC6 hydrophobic segment from 17 to 21 amino
acids, which resulted in re-targeting to the Golgi complex. A further
increase in length to 26 amino acids allowed this modified protein to
traverse the secretory pathway and gain expression at the plasma
membrane. These findings are consistent with models in which, in the
absence of dominant cytosolic or lumenal targeting determinants,
proteins may be sorted within the secretory pathway based on
interactions between their transmembrane domains and the surrounding
lipid bilayer.
Fundamental to understanding the secretory pathway is an
elucidation of the molecular basis by which organelles of this system are formed, and a determination of the means by which steady state levels of resident proteins and lipid are maintained. As the default pathway for the vectorial flow of proteins leads from the
ER1 to the plasma membrane (1, 2),
mechanisms are necessary to retain resident proteins within
intracellular organelles. Several specific signals have been identified
for the retention/retrieval of ER proteins. Retrieval from the Golgi of
soluble ER resident proteins is mediated by recognition of the
tetra-amino acid sequence "KDEL" by a specific receptor (3). For
transmembrane proteins, intracytoplasmic sequences play a role in ER
retention/retrieval. A di-lysine motif at the C terminus of type I
proteins allows for retrieval from the Golgi to the ER in a
coatomer-dependent manner (4-7); a di-arginine N-terminal
motif may play an analogous role for type II proteins (8), and a
tyrosine-based motif also functions as a cytoplasmic ER retention
signal (9). Proteins can also be indirectly retained by interactions
with ER resident proteins. For example, lumenal chaperones including
BiP, calnexin, and calreticulin interact with newly synthesized
proteins in the ER lumen and mediate transient or stable retention of
proteins that are devoid of intrinsic ER retention/retrieval sequences (10).
UBC6 is a 250-amino acid ER resident protein that has no known ER
retention/retrieval signal (11). This membrane-anchored Saccharomyces cerevisiae ubiquitin-conjugating enzyme is
implicated in the degradation of a transcriptional repressor (MAT A cDNA
encoding UBC6 in pTX8 was a gift from S. Jentsch (University of
Heidelberg). A 1.02-kilobase fragment was subcloned into
pGEM7z+ (Promega, Madison WI) and subsequently transferred
to pSVL (Pharmacia Biotech Inc.). UBC6 with an N-terminal myc epitope
tag and four-amino acid linker (see Fig. 1) was generated by PCR using
appropriate oligonucleotides2 and UBC6 in
pGEM7z+ as template (Fig. 1). PCR was carried out with
conditions of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for
2 min for 30 cycles. The product was cloned into pGEM7z+,
and subcloned into pSVL from XhoI to BamHI
(pSVL-mUBC6). In a similar fashion, UBC6 with a C-terminal
myc tag was generated by PCR and subcloned into pSVL. To generate
variants in the C terminus of mUBC6, an oligonucleotide
corresponding to bases 517-540 of UBC6 (U517-40) was paired with
antisense (3
Myc-tagged ZAP-70 was from L. Samelson and R. Wange (National
Institutes of Health) (17). Tac-E19 has been previously described (18);
plasmids encoding mannosidase II and the interleukin-2 receptor COS-7 cells (American Tissue Culture
Collection, number CRL1651, Rockville, MD) were maintained in
Dulbecco's modified Eagle's medium (Biofluids, Rockville, MD)
supplemented with 8% fetal bovine serum (complete medium). Hybridomas
secreting monoclonal antibodies 9E10 (anti-myc) (21) and 7G7
(anti-interleukin-2 Immunofluorescence on
transfected COS-7 cells was carried out as described (26) using
secondary antibodies obtained from Jackson ImmunoResearch (West Grove,
PA) conjugated to indocarbocyanine (Cy3), fluorescein, or rhodamine.
For co-localization experiments, mUBC6 constructs were
co-transfected with known markers. After incubation with primary mouse
and rabbit antibodies, double staining was carried out with
fluorescein-labeled anti-mouse and rhodamine-labeled anti-rabbit
secondary antibodies. Transfection efficiencies were generally
20-30%, in all experiments over 200 transfected cells were examined
for each field shown. For time course studies (Fig. 7), cells were
transfected by electroporation using a Bio-Rad Gene Pulsar, with
conditions of 240 V and 500 microfarads with 5 × 106
cells in 0.2 ml of complete media. Electroporated cells were plated on
coverslips and processed at the indicated times. Brefeldin A (Epicentre
Technologies, Madison, WI) was used at a final concentration of 1 µg/ml.
For metabolic labeling
studies, cells were transfected with 20 µg of DNA by calcium
phosphate precipitation (27). After 24 h, cells were removed from
plates using Versene (Biofluids) and labeled in 1 ml of methionine-free
complete medium containing 0.3 mCi/ml of [35S]methionine
(Tran35S-label, ICN Radiochemicals, Irvine, CA) for 30 min
at 37 °C. Cells were then washed and resuspended in complete medium.
At designated time points cells were collected and washed three times in ice-cold PBS. Detergent lysis of cell pellets, immunoprecipitation, and SDS-PAGE have all been described (28). Gels were fixed followed by
impregnation with Enlightning (DuPont NEN) and autoradiography at
For analysis of membrane and cytosolic fractions, transfected COS-7
cells were resuspended in 2 ml of 30% normal tonicity PBS supplemented
with protease inhibitors (28). After 10 min at 4 °C, cells were
disrupted with 40 strokes of a Dounce homogenizer, and tonicity was
immediately restored with hypertonic PBS. After removal of nuclei and
unbroken cells at 300 × g, membranes were pelleted at
100,000 × g. Membranes were solubilized in Triton X-100 containing buffer (28), and membrane and cytosolic samples were
immunoprecipitated, resolved on SDS-PAGE, transferred to nitrocellulose, and subjected to Western blotting with biotin-labeled 9E10 followed by detection using streptavidin-horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) and enhanced chemiluminescence (Amersham Life Sciences).
Following electroporation with 25 µg of plasmid (as above), 107 COS-7 cells were plated
into duplicate 100-mm dishes. After 48 h, plates were rinsed twice
with Buffer A (0.25 M sucrose, 10 mM
triethanolamine, 1 mM EDTA, pH 7.4) and harvested by gentle scraping into 5 ml of homogenization buffer (85% Buffer A (v/v) and
15% of Buffer B (10 mM Tris, pH 7.4, 5 mM KCl,
1 mM EDTA, 128 mM NaCl)). Cells were spun at
300 × g and resuspended in 0.3 ml of homogenization
buffer plus protease inhibitors (28), followed by homogenization at
4 °C by 12 passages through a 25-gauge needle attached to a 1-ml
syringe. Post-nuclear supernatants were prepared by two consecutive
centrifugations at 1000 × g. Supernatants were loaded
on pre-formed 0-26% Optiprep (Iodixanol), linear gradients that had
been pre-cooled to 4 °C. Gradients were spun for 115 min at 41,000 rpm at 4 °C in a SW41 rotor in a Beckman ultracentrifuge. Twenty
fractions (600 µl each) were recovered by bottom piercing using a
Beckman Fraction Recovery System. For immunoblotting, fractions were
diluted to 1.2 ml with Buffer B, and membranes were pelleted in a
Beckman High Speed Microfuge at 4 °C in a TLA45 rotor at
125,000 × g for 60 min. Pellets dissolved in SDS-PAGE sample buffer were subject to electrophoresis and immunoblotting.
Gradients were formed by underlayering 6 ml of 26% Optiprep solution
(26% Iodixanol (Nycomed, Oslo, Norway) in Buffer B) under 6.5 ml of
Buffer B containing added protease inhibitors in SW41 tubes (Beckman)
and inserting into a Gradient Master (Biocomp Instruments, Fredericton,
Canada) and rotating at 15 rpm for 2.22 min at 1.5° above the
horizontal angle. The resultant gradient was from bottom to top 26 to
0% Iodixanol. Cell surface biotinylation (29) and the
galactosyltransferase assay (30) were carried out as described.
To determine the subcellular location of UBC6 when
expressed in mammalian cells, a construct was generated encoding UBC6
with a myc-epitope tag at its N terminus (mUBC6) (Fig.
1). This tag allows for recognition by an
anti-myc-peptide monoclonal antibody, 9E10 (21). When mUBC6
was transiently expressed in COS-7 cells and examined by
immunofluorescence microscopy using 9E10 as the primary antibody, a
lacy pattern characteristic of the ER was seen (Fig.
2A). Untagged UBC6 (assessed using anti-UBC6
rabbit sera) and C-terminal myc-tagged UBC6 exhibited similar ER
patterns (not shown). Since interpretation of results with the
N-terminal epitope tag are not complicated by issues related to the
introduction of a lumenal domain, mUBC6 was used as the
basis for the construction of additional epitope-tagged constructs
(Fig. 1).
As UBC6 has no intralumenal domain, cytosolic and/or transmembrane
domains (TMDs) must play an essential role in its localization. To
evaluate this, the TMD of the human transferrin receptor (TfR) (31) was
substituted for the native TMD of UBC6
(mUBC6TfR) (Fig. 1). Structurally, the TfR is a
typical type II protein, normally targeted to the plasma membrane where
it undergoes endocytosis and re-cycling to the cell surface (32). When
cells expressing mUBC6TfR were assessed by
immunofluorescence microscopy, the pattern found was dramatically
different from that of mUBC6, as
mUBC6TfR was prominently expressed at the plasma
membrane, although staining of intracellular membranes was still
apparent (Fig. 2B). This indicates that a heterologous TMD
is sufficient to re-target UBC6 to the plasma membrane and suggests
that the TMD plays a dominant role in determining the subcellular
location of UBC6. To ascertain whether the UBC6 TMD is sufficient to
direct a heterologous protein to the ER, a construct was generated
encoding the UBC6 TMD appended to the C terminus of myc-tagged green
fluorescence protein (16) (mGFPUBC6). Staining
with 9E10 revealed a pattern consistent with ER localization (Fig.
2C). Thus, it would appear that the TMD of UBC6 is necessary for targeting of UBC6 to the ER and also capable of targeting a
heterologous protein to ER membranes.
The TMD of UBC6 is only 17 amino acids in length, whereas
the TfR has a predicted membrane-spanning segment of 28 amino acids. To
determine if TMD length plays a role in the ER location of UBC6, the
TMD of mUBC6 was lengthened by the insertion of four additional hydrophobic amino acids (VAVA) prior to the C-terminal lysine, for a total of 21 transmembrane residues (Fig. 1). When this
modified molecule, mUBC6TM21 (Fig.
2D), was compared with mUBC6, a marked change in
distribution was observed, with mUBC6TM21 found
mainly in a discrete perinuclear region characteristic of the Golgi
complex.
It has been proposed that the generally shorter TMDs of resident Golgi
proteins relative to their plasma membrane counterparts is, at least in
part, responsible for their subcellular localization (33). To address
whether mUBC6TM21 could be retargeted from the
Golgi to the plasma membrane by further lengthening of the TMD, its
hydrophobic segment was increased by five more amino acids (LLVAL), for
a total of 26 transmembrane residues (Fig. 1). When expressed
transiently, this protein, mUBC6TM26, exhibited
a pattern similar to that seen with mUBC6TfR,
consistent with expression both at the plasma membrane and in intracellular compartments (Fig. 2E). To ensure that this
plasma membrane expression was not due to the incidental generation of a plasma membrane targeting sequence, a second construct encoding a
different five amino acid addition (ILALV) was assessed
(mUBC6TM26 The distributions of the various mUBC6 constructs were
confirmed by co-localization studies with known ER, Golgi, and plasma membrane markers (Fig. 3). mUBC6, bearing the
wild type TMD, exhibits a pattern almost indistinguishable from the
ER-retained protein, Tac-E19 (Fig. 3, A and B);
mUBC6TM21 co-localizes with a Golgi marker,
mannosidase II (Fig. 3C and D); and finally, the
staining found with mUBC6TM26 is strikingly
similar to that seen with the Tac antigen (the interleukin-2 receptor
To confirm that all of the UBC6-based proteins are membrane-bound,
cytosolic and membrane fractions were prepared from cells expressing
epitope-tagged UBC6 constructs and immunoprecipitated with 9E10 (Fig.
5). The mUBC6-derived proteins were found
almost exclusively in the membrane fractions. In contrast, a myc-tagged non-transmembrane tyrosine kinase, ZAP-70 (34), was found largely in
the cytosolic fraction. To determine if the membrane orientation of
UBC6 is maintained when the transmembrane domain is lengthened to 26 amino acids, cells that were fixed and permeabilized prior to staining
with 9E10 were compared with those that were not permeabilized. Permeabilization and fixation was required for recognition of mUBC6TM26 by 9E10, while Tac was recognized by
an antibody directed against its ectodomain regardless of
permeabilization (not shown). Thus, lengthening the transmembrane
domain of UBC6, while altering its subcellular distribution, does not
change its membrane association or its orientation.
The most straightforward explanation for these findings is that the
length of the hydrophobic transmembrane segment is responsible for
determining the subcellular location of UBC6. An alternative possibility is that proteins bearing the wild type TMD are rapidly degraded after synthesis and therefore only seen in the ER, whereas those with heterologous or modified TMDs are more stable and
consequently visualized at later stages during their progression
through the secretory pathway. To address this, the relative half-lives
of the mUBC6 constructs were examined by pulse-chase
metabolic labeling using [35S]methionine. As shown (Fig.
6), mUBC6, mUBC6TM21,
mUBC6TM26, and mUBCTfR
were all stable for at least 6 h. In contrast,
mUBC6TM14, which has only a 14-amino acid
hydrophobic stretch, was lost with a t1/2 of less
than 2 h. These results establish that differences in
compartmental distribution among variants of mUBC with
different length TMDs cannot be accounted for by differences in their
relative stabilities.
Most integral membrane proteins of the secretory pathway
gain entry into ER membranes via a co-translational,
SRP-dependent mechanism and are then either retained in the
ER or transported to their final destinations (14, 35, 36). While
substantially less is known regarding the post-translational insertion
of C-terminal anchored proteins (13), there are at least two examples
where members of this family appear to be inserted into ER membranes (37, 38). The distribution of mUBC6TM26, as
assessed by immunofluorescence microscopy and subcellular fractionation
(Figs. 3 and 4), is consistent with the idea that this protein reaches
the plasma membrane after first being inserted into the ER. To evaluate
this further, the time course of plasma membrane expression of
mUBC6TM26 was assessed. Cells transfected by
electroporation with mUBC6TM26 were examined
5 h after transfection (Fig. 7A) or
incubated for an additional 5 h either without or with the
addition of brefeldin A (Fig. 7, B and C).
Brefeldin A is a fungal product that disrupts the Golgi complex,
preventing newly synthesized proteins from reaching the plasma membrane
(39-41). As shown, at 5 h after transfection, the pattern seen
with mUBC6TM26 is consistent with distribution
in the ER, with perhaps some Golgi staining as well. Incubation for an
additional 5 h results in the development of plasma membrane
staining that was not visible at 5 h and which was blocked by
brefeldin A. Similar findings were obtained with the type I plasma
membrane protein Tac (not shown). These observations are consistent
with the notion that C-terminal anchored proteins with long
transmembrane domains, like proteins that gain access to the secretory
pathway in an SRP-dependent fashion, are inserted into the
ER and then sorted to their final destinations.
For most transmembrane proteins, hydrophobic signal peptides
facilitate the SRP-dependent co-translational insertion of
nascent proteins into the ER membrane (14, 35, 36). Type IV (C-terminal anchored) transmembrane proteins are an exception, as they are introduced into membranes in a post-translational, SRP-independent manner. Little is known regarding the mechanisms responsible for membrane insertion and intracellular localization of type IV proteins; however, their varied patterns of expression demonstrates that they
encode sufficient information to confer targeting to specific intracellular addresses (13).
UBC6 is a type IV ER membrane protein that contains no canonical ER
retention/retrieval sequences. Substitution of a heterologous 28-amino
acid TMD re-targets UBC6 to the plasma membrane, whereas ER
localization is imparted to a soluble protein by addition of the wild
type (17-amino acid) UBC6 TMD. Lengthening of the wild type UBC6 TMD to
21 amino acids allows for movement to the Golgi complex and a further
increase to 26 amino acids facilitates traversal of the secretory
pathway, leading to expression at the plasma membrane. Thus, it appears
that for this type IV membrane protein, the length of the TMD plays a
crucial role in targeting within the secretory pathway.
TMDs have been implicated in ER retention in other systems. Unassembled
T cell antigen receptor TMDs play major roles in retention of proteins within the Golgi stack,
as exemplified by galactosyltransferase, sialyltransferase, the avian
coronavirus M protein, and other resident Golgi proteins (48-55).
Whereas the range of TMD properties responsible for Golgi retention
remains to be established, in general Golgi proteins have substantially
shorter TMDs than their cell surface counterparts. This has led to the
hypothesis that lipid composition-dependent differences in
thickness and deformability between the lipid bilayers of the Golgi
complex and those of the plasma membrane play a determining role in
protein sorting (33). Since cholesterol is known to increase membrane
thickness and to decrease deformability, and because its concentration
in lipid bilayers increases as one proceeds outward through the
secretory pathway (56), it has been implicated as being of consequence
in "lipid-based" sorting between the Golgi and the plasma membrane
(33, 55). In a lipid-based sorting model, proteins partition between
the Golgi complex and the plasma membrane based on energetics; shorter
hydrophobic segments preferentially distribute to the Golgi and longer
ones to the thicker plasma membrane bilayer. The concept that TMD
length determines distribution between the Golgi and the plasma
membrane has recently been confirmed experimentally for both a Golgi
and a plasma membrane protein (55).
The change in distribution associated with incremental increases in the
length of the UBC6 TMD suggests that a lipid-based sorting mechanism
may be operative not only between the Golgi and the plasma membrane but
also between the cholesterol-poor, thinner/more deformable lipid
bilayers of the ER and those of the Golgi complex (56, 57). Based on
correlations between charged TMD residues and ER retention, it seems
likely that it is not only TMD length but also overall hydrophobicity
that determines the ability of proteins to move out of the ER. ER
retention of UBC6 and other proteins would represent the extreme
situation where the TMDs are long enough for membrane insertion but too short and/or hydrophilic to allow these proteins to exit the ER and
move to thicker/more rigid membranes. Lipid-based sorting does not
preclude a role for TMD oligomerization in determining protein
distribution. In fact, it has been suggested that oligomerization may
improve the efficiency with which some proteins are retained in
cisternae of the Golgi complex through TMD-mediated interactions (58).
In other instances, such as with the charge-containing TMDs of the
multi-subunit T cell antigen receptor, associations between TMDs of
different subunits having opposing charges could affect the physical
characteristics displayed to the hydrophobic milieu and thus facilitate
the egress of assembled receptors from the ER, leading to their
eventual expression at the cell surface (42).
The generation and maintenance of distinct protein populations among
organelles of the secretory pathway is accomplished, at least in part,
by specificity at the levels of vesicular transport and protein
retention/retrieval. However, the proteins that form the bases for
these targeting mechanisms must themselves be directed to specific
compartments. Sorting mechanisms based on TMD length and hydrophobicity
represent an efficient means for establishing such distributions as a
consequence of physical interactions between TMDs and their surrounding
lipid environment. In fact, the recent findings that several SNARE
proteins are C-terminal anchored proteins with relatively short TMDs
(37, 59-61) may be indicative of a primary role for TMDs in
establishing these distributions. Our findings, placed in the context
of the existing literature, make it reasonable to surmise that a
gradient in the physical characteristics of the lipid bilayer exists
within the secretory pathway, such that, in the absence of dominant
lumenal or cytosolic associations, proteins distribute based on
interactions between TMDs and the surrounding lipid environment. As
C-terminal anchored proteins contain no lumenal domains, they represent
potentially useful tools to further evaluate protein sorting among
organelles of the secretory pathway.
Laboratory of Immune Cell Biology,
2)
and of a temperature-sensitive mutant of the ER translocator (Sec 61).
This has led to speculation that UBC6, and perhaps mammalian homologues, plays a role in ubiquitin-mediated degradation of ER
proteins (11, 12). UBC6 is a member of the C-terminal anchored, or type
IV, class of transmembrane proteins. These proteins are characterized
by hydrophobic segments close to, or at, their C termini, precluding
the co-translational SRP-dependent membrane insertion
characteristic of most transmembrane proteins (13, 14). In this study
we evaluate the basis for ER retention of UBC6 and establish that the
C-terminal hydrophobic membrane anchor plays a dominant role in
determining the localization of UBC6 within the secretory pathway.
DNA Recombinant Procedures and Transfections
) oligonucleotides encoding the desired C-terminal
modifications in the PCR (Fig. 1). Products were cut with
XbaI and BamHI and cloned into
pSVL-mUBC6 that had been cut with XbaI and
BamHI. A construct encoding mUBC6TfR
was generated by a two-step PCR method (15). The final product corresponds to the cytoplasmic coding region of mUBC6 linked
to amino acids 61-90 of the TfR (Fig. 1). Green fluorescence protein (16) with a recognition site for 9E10 at the N terminus and the
C-terminal 18 amino acids of UBC6 at its C terminus
(mGFPUBC6) was also generated by two-step
PCR.
Fig. 1.
Modifications of UBC6. Myc-tagged UBC6
(mUBC6) and derivatives thereof with altered TMDs are
represented schematically. The sequence recognized by the monoclonal
antibody 9E10 is boxed, and the tetra amino acid sequence
"ELGV" serves as a linker to the body of UBC6.
[View Larger Version of this Image (26K GIF file)]
subunit (Tac) were from N. Cole (National Institutes of Health) (19),
and W. Leonard (National Institutes of Health) (20),
respectively.
subunit) (22) were obtained from the American
Tissue Culture Collection and from D. Nelson (National Institutes of
Health), respectively. Polyclonal rabbit antisera raised against UBC6
(11), anti-interleukin-2 receptor
subunit (Tac) (23), mannosidase
II (24), and ribophorin I (25) were from T. Sommer and S. Jentsch
(University of Heidelberg), W. Leonard (National Institutes of Health),
K. Moremen (Whitehead Institute), and G. Kreibich (New York
University).
Fig. 7.
mUBC6TM26 reaches the plasma
membrane by traversing the secretory pathway. Cells transfected
with mUBC6TM26 by electroporation were analyzed
by immunofluorescence at 5 h after transfection (A) or
10 h after transfection (B). C, brefeldin A
(BFA) (1 µg/ml) was added at 5 h after transfection,
and cells were analyzed after a total of 10 h. Shown are
representative fields from over 300 transfected cells that were
analyzed.
[View Larger Version of this Image (57K GIF file)]
70 °C.
The Transmembrane Domain of UBC6 Determines Its Subcellular
Location
Fig. 2.
The TMD of UBC6 determines its subcellular
location. Constructs encoding mUBC6 (A),
mUBC6TfR (B),
mGFPUBC6 (C),
mUBC6TM21 (D),
mUBC6TM26 (E), or
mUBC6TM26 (F) were transiently
expressed in COS-7 cells by calcium phosphate precipitation. Cells were
analyzed by immunofluorescence microscopy 40 h after transfection
using the monoclonal anti-myc peptide 9E10, followed by Cy3-conjugated
donkey anti-mouse IgG. Shown are representative fields from multiple
independent transfections. Transfection efficiencies were generally
20-30%.
[View Larger Version of this Image (82K GIF file)]
). As is evident, the pattern found
with mUBC6TM26
(Fig. 2F) is similar
to that found with mUBC6TM26.
subunit) (Fig. 3, E and F), a protein known
to be expressed on the cell surface (18, 20, 24). The TMD-mediated
re-targeting of mUBC6 was further evaluated by subcellular
fractionation using Iodixanol-based linear density gradients. In these
studies, mUBC6 was found predominantly in the denser
fractions of the gradients, where it co-migrated with an endogenous ER
protein, ribophorin I (Fig. 4, compare panels A and B). mUBC6TM26, on the
other hand, exhibited a more heterogeneous distribution, with over 60%
of the recovered protein in less dense (post-ER) fractions, consistent
with trafficking of this protein from the ER through the Golgi to the
plasma membrane.
Fig. 3.
Co-localization with known cellular
markers. COS-7 cells were co-transfected with constructs encoding
either mUBC6 and a known ER marker, Tac-E19
(A and B); mUBC6TM21 and a
known Golgi marker, mannosidase II (C and D); or
with mUBC6TM26 and Tac (a cell
surface protein) (E and F). Fixed and
permeabilized cells were incubated with 9E10, which recognizes the
epitope-tagged mUBC6, mUBC6TM21, and
mUBC6TM26 and with rabbit polyclonal antisera
directed against each of the appropriate markers. Cells were then
incubated with fluorescein-conjugated donkey anti-mouse IgG and
rhodamine-conjugated donkey anti-rabbit IgG. The subcellular locations
of the mUBC6-derived proteins are demonstrated in A,
C, and E using 488 nM excitation
(fluorescein), whereas B, D, and F are the
corresponding fields evaluated for the known markers using 568 nM excitation (rhodamine). The fields shown are
representative of over 300 transfected cells analyzed in two
independent experiments.
[View Larger Version of this Image (89K GIF file)]
Fig. 4.
Subcellular fractionation of mUBC6 and
mUBCTM26. COS-7 transfected with either
mUBC6 () or mUBCTM26 (
) were
separated by differential centrifugation as described under
"Materials and Methods," and fractions were analyzed by immunoblotting with 9E10 (A). Shown for comparison are the
distribution of ER (ribophorin I,
), Golgi (galactosyltransferase,
), and plasma membrane (biotinylated cell surface proteins,
)
markers (B). The densities at the top and
bottom of the gradient are indicated. For each distribution,
results are presented normalized to the maximum value.
[View Larger Version of this Image (24K GIF file)]
Fig. 5.
Localization of mUBC6 proteins to membrane
fractions. Membrane (M) (lanes 1, 3, 5, 7, and 9) and cytosolic (C) (lanes 2, 4, 6, 8, and 10) fractions of COS-7 cells transfected with either mUBC6 (lanes 1 and 2),
mUBC6TM21 (lanes 3 and 4),
mUBC6TM26 (lanes 5 and 6),
mUBC6TfR (lanes 7 and 8),
or mZAP-70 (lanes 9 and 10) (17) were
immunoprecipitated with 9E10 and resolved on 12% SDS-PAGE followed by
immunoblotting with biotinylated 9E10 and developed with
streptavidin-conjugated horseradish peroxidase and enhanced
chemiluminescence (see "Materials and Methods" for details). The
small amount of ZAP-70 seen in the membrane fraction (lane
9) may be a consequence of SH2-mediated associations with membrane
proteins.
[View Larger Version of this Image (23K GIF file)]
Fig. 6.
Pulse-chase analysis of mUBC-derived
proteins. Cells transfected with mUBC6 (lanes
1-3), mUBC6TM21 (lanes 4-6),
mUBC6TM26 (lanes 7-9),
mUBC6TfR (lanes 10-12), or
mUBC6TM14 (lanes 13-15) were pulsed
for 30 min with [35S]methionine and chased for either 0, 2, or 6 h in complete medium as indicated, followed by
immunoprecipitation with 9E10 and resolution on 12% SDS-PAGE.
[View Larger Version of this Image (20K GIF file)]
and
subunits are retained in the ER, at
least in part, as a consequence of charged TMD amino acids (42). For a
plasma membrane protein with a 19-amino acid TMD, introduction of
charged residues into the hydrophobic segment leads to ER retention
and, depending on the placement of the charge, to degradation (43).
Similarly, for another cell surface-expressed chimera that has a charge
within its TMD, shortening the predicted TMD from 23 to 17 amino acids
leads to ER retention (44). Interestingly, the KDEL receptor contains
an aspartic acid in one of its TMDs that is crucial for ER retrieval
(45). The 20-amino acid TMD of microsomal cytochrome P450 targets to
the ER, contains no charged residues, but does include several
hydrophilic amino acids (46), and while this manuscript was being
prepared, a study was published demonstrating that lengthening the
17-amino acid TMD of cytochrome b5 by 5 amino
acids resulted in a re-distribution from the ER to the cell surface
(47).
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a fellowship of the Boehringer Ingelheim Fonds,
Germany.
To whom correspondence should be addressed: Bldg. 10, Rm. 1B34
National Institutes of Health, 9000 Rockville Pike, Bethesda, MD
20892-1152. Tel.: 301-496-3557; Fax: 301-402-4844; E-mail: amw{at}nih.gov.
1
The abbreviations used are: ER, endoplasmic
reticulum; SRP, signal recognition protein; PBS, phosphate-buffered
saline; PAGE, polyacrylamide gel electrophoresis; TfR, transferrin
receptor; TMD, transmembrane domain; PCR, polymerase chain
reaction.
2
Sequences of oligonucleotides are available on
request.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.