(Received for publication, September 11, 1995; and in revised form, December 18, 1995)
From the
An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J.(1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.
The endomembranes participating in the early stages of protein
maturation and transport consist of the endoplasmic reticulum (ER), ()the cis-Golgi cisternae and tubulo-vesicular
elements at the interface between these organelles (Palade, 1975).
After their translocation across the membrane of the rough ER, newly
synthesized secretory and membrane proteins undergo folding and
post-translational modification reactions, before becoming competent to
be transported out of the ER (Helenius et al., 1992; Gething
and Sambrook, 1992; Helenius, 1994). Transport along this pathway
occurs via an intermediate compartment which could originally be
visualized in Semliki Forest virus-infected cells using low temperature
as a reversible transport arrest (Saraste and Kuismanen, 1984).
Identification and localization of two marker proteins, rat p58
(Saraste et al., 1987) and human p53 (Schweizer et
al.(1988); renamed ERGIC-53, Schindler et al.(1993)), has
provided additional information of the structural complexity of this
compartment. The tubulo-vesicular elements, to which these proteins
concentrate (Schweitzer et al., 1991; Lahtinen et
al., 1992), have been shown to be involved in the transport of
viral glycoproteins to the Golgi complex (Schweitzer et al.,
1990; Saraste and Svensson, 1991; Plutner et al., 1992).
The removal of membrane from the ER, that accompanies the formation of vesicular carriers, is balanced by the counterflow of lipids and retrieval of membrane proteins from the Golgi (Wieland et al., 1987). This retrograde pathway serves as a mechanism to return escaped, KDEL-containing, resident proteins back to the ER (Pelham, 1988; Dean and Pelham, 1990) and maintain the localization of either KKXX- or KXKXX-containing ER membrane proteins by retrieval (Nilsson et al., 1989; Jackson et al., 1990; Jackson et al., 1993; Townsley and Pelham, 1994; Gaynor et al., 1994).
Studies using in vitro assays and yeast secretion mutants have identified several
cytosolic components needed for vesicle budding, transport, and fusion
(reviewed by Rothman, 1994). Coatomer is a complex of seven proteins
(-,
-,
`-,
-,
-,
-, and
-COP), which
assemble on membranes in an ADP-ribosylation factor-dependent manner
forming a cytoplasmic coat (Serafini et al., 1991; Waters et al., 1992; Orci et al., 1993; Ostermann et
al., 1993). Coatomers are involved both in intra-Golgi (Rothman
and Orci, 1992) as well as ER to Golgi transport (Pepperkok et
al., 1993; Peter et al., 1993). Recently, coatomer
subunits and double-lysine motifs have been shown to interact in
vitro (Cosson and Letourneur, 1994). The functional significance
of this interaction in membrane dynamics between the ER and Golgi was
revealed by genetic studies in yeast (Letourneur et al., 1994)
showing that several coatomer subunits were essential for protein
retrieval.
As a first stage in trying to understand both the function and the molecular mechanism for trafficking of p58 we have cloned and expressed its cDNA and developed peptide antibodies against different domains of the protein. We report here that p58 is the rat homolog of the human ERGIC-53/MR60 protein (Schindler et al., 1993; Arar et al., 1995), which has been suggested to function as a membrane bound lectin in the early secretory pathway (Fiedler and Simons, 1994; Arar et al., 1995). Furthermore, immunolocalization using the anti-tail peptide antibodies detected p58 only in the Golgi complex, suggesting masking of the tail in earlier compartments.
For metabolic labeling, the cells were first preincubated with methionine- and cysteine-free medium containing 10% dialyzed fetal calf serum for 1 h and then labeled with Pro-Mix (500 µCi/ml).
Due to the relatively high background observed in immunofluorescence staining, ImmunoPure Ag/AB Immobilization Kit #2 (Pierce) was used for affinity purification of the anti-tail antibodies according to the manufacturer's instructions.
To introduce either a c-Myc- or HA-epitope at the N terminus (alanine 42) an unique NarI-restriction site was used. Sense and antisense oligonucleotides encoding amino acids GEQKLISEEDLG (c-Myc, bold) or GYPYDVPDYAG (HA, bold) and containing NarI sites were synthesized, annealed, and ligated.
For transfections with both the wild type and c-Myc- or HA-tagged p58, the cDNAs were transferred into the pSFV1 vector (Liljeström and Garoff, 1991).
A X. laevis liver UNI-ZAP XR cDNA library was
screened with a 1.3-kilobase rat p58 cDNA probe. Hybridizations were
carried out at 42 °C in 4 SSPE, 25% formamide, 5
Denhardt's solution, and 100 µg/ml salmon sperm DNA.
Nucleotide sequences were determined from both strands by using the Sequenase version 2.0 Sequencing Kit (U. S. Biochemical Corp., Amersham).
In vitro translations using rabbit reticulocyte lysate and canine pancreatic microsomal membranes were performed as described by the manufacturer (Promega). Proteinase K digestions were carried out as described earlier (Lahtinen et al., 1992).
Figure 3:
In vitro translation of p58 cDNA. A, translation of p58 cDNA in the absence (lane 2)
and presence (lane 3) of microsomes and a control
precipitation from S-labeled BHK cells (lane 1).
Translation of c-Myc- and HA-tagged p58 in the absence and presence of
microsomes (lanes 4-7). B, alkaline extraction
of microsomes followed by centrifugation showing the recovery of
translated p58 in the pellet (lane 1) and its absence in the
supernatant (lane 2). C, proteinase K digestion of
microsomes. Precipitations of p58 from untreated microsomes with
anti-tail (lane 1) and anti-p58 (lane 2) antibodies,
and from digested microsomes with anti-tail (lane 3) and
anti-p58 (lane 4) antibodies. Positions of molecular weight
markers are shown to the left.
Figure 1: Nucleotide sequence of p58 and the deduced amino acid sequence. The putative N-terminal signal sequence (medium bar), the four microsequenced peptides (thin bar), and the predicted C-terminal transmembrane segment (thick bar) are underlined.
The p58 cDNA has an open reading frame encoding 517 amino acid residues (Fig. 1). The N terminus is predicted to contain a 30-residue long and cleavable signal sequence (von Heijne, 1985), followed by a lumenal domain. Based on the hydropathy profile (Kyte and Doolittle, 1982; data not shown) and the position of the flanking charged residues, a 18-residue long hydrophobic stretch close to the C terminus is likely to form a transmembrane domain, followed by a 12-amino acid long cytoplasmic tail. Mature p58 is predicted to have molecular mass of 54,755 daltons and pI of 5.9. In agreement with previous biochemical data (Lahtinen et al., 1992), no consensus sites for N-glycosylation were found.
Figure 2: Comparison of the rat p58, human ERGIC-53, and frog p58 amino acid sequences. Rat and human sequences share 89% identity (94% similarity), and rat and frog sequences 71% identity (85% similarity) indicating high evolutionary conservation of the protein.
To obtain further information of the degree on evolutionary conservation of p58, we screened a X. laevis UNI-ZAP XR cDNA library with a 1.3-kilobase rat p58 cDNA fragment. Two positive overlapping clones were isolated having a long open reading frame and a stop codon, but both lacking the start codon. The Xenopus protein was estimated to have the same size as the rat p58 since anti-p58 antibodies recognized a protein co-migrating with the rat p58 in immunoblots of frog liver homogenates (data not shown). The C-terminal 420-amino acid sequence was 71% identical (85% similar) to the rat sequence (Fig. 2).
When membranes were subjected to extraction with sodium carbonate (pH 11.3) followed by centrifugation, the in vitro translated p58 was almost quantitatively recovered in the resulting pellet (Fig. 3B), confirming proper membrane integration.
Previous experiments have shown that p58 remained largely protected in microsomes after protease treatments (Lahtinen et al., 1992). To probe the membrane topology of in vitro synthesized p58, anti-tail peptide antibodies (see below) were used. Both anti-tail and polyclonal anti-p58 antibodies precipitated the intact protein (Fig. 3C, lanes 1 and 2). After digestion with proteinase K, anti-tail antibodies failed to precipitate the protein, indicating that the short tail was accessible to the enzyme (lane 3), whereas anti-p58 antibodies precipitated a slightly faster migrating protected form (lane 4). Together these results show that p58 is a type I integral membrane protein, having the bulk of the protein on the lumenal side and a short C-terminal cytoplasmic tail in agreement with the topology deduced from the amino acid sequence (see above).
Figure 4:
Specificity of the peptide antibodies.
Anti-lumenal p58 antibodies and anti-tail p58 antibodies (lanes 1 and 3, respectively) specifically recognize a 58-kDa
protein in immunoblots of rat pancreatic microsomes. Immunoprecipitates
from cell lysates from S-labeled BHK cells with
anti-lumenal (lane 2) and anti-tail (lane 4)
antibodies. The high molecular weight bands probably represent
aggregated p58.
When tested by immunofluorescence a distinct difference in the staining patterns obtained with the two peptide antibodies was observed in BHK cells. The staining with the anti-lumenal antibodies was similar to the staining obtained with the previously described affinity-purified anti-p58 antibodies (Saraste and Svensson, 1991); the Golgi region as well as vesicular elements concentrated in the Golgi area, but also scattered throughout the cell, were seen (Fig. 5, panels A and B). In contrast, the anti-tail antibodies stained a perinuclear, often ring-shaped structure typical of the Golgi complex (panel C). No scattered punctate elements could be visualized even in transfected cells having higher expression levels of p58 (data not shown). This suggests that in the vesicular elements the epitope(s) in the C-terminal tail may be hidden and therefore not accessible to the anti-tail antibodies.
Figure 5: Immunofluorescence staining of endogenous p58 in BHK cells. Staining with anti-p58 antibodies (A), anti-lumenal p58 antibodies (B), and anti-tail p58 antibodies (C) visualized by FITC-coupled secondary antibodies.
For in vivo expression, both the c-Myc- and HA-tagged p58 cDNAs were cloned into a Semliki Forest virus-based pSFV1-vector (Liljeström and Garoff, 1991), the DNA was transcribed in vitro, and transfected into BHK cells by lipofection. Four hours post-transfection, cells were treated for 1 h with 50 µg/ml cycloheximide and then fixed and processed for immunofluorescence. Staining with anti-c-Myc (Fig. 6, panel A) antibodies showed a punctate, Golgi-concentrated pattern resembling the distribution of endogenous p58. Double-staining with anti-mannosidase II antibodies (panel B) showed overlapping distributions of the two antigens in the Golgi area as previously observed both in BHK and normal rat kidney cells (Saraste and Svensson, 1991). In cells having a higher expression level, the protein could also be clearly detected in reticular ER membranes. This is in good agreement with previous immunolocalization studies and the quantitation of p58 in subcellular fractions (Saraste and Svensson, 1991; Lahtinen et al., 1992). The same distribution was observed with the HA-tagged p58 (data not shown). In addition, c-Myc-tagged p58 was shown to form both homodimers and oligomers as well as heterodimers and oligomers with the endogenous protein indicating that the oligomerization (Lahtinen et al., 1992) was not affected by the introduction of a foreign peptide sequence at the N terminus (not shown).
Figure 6: Expression of c-Myc-tagged p58. c-Myc-tagged p58 was expressed by using the Semliki Forest virus system. Immunofluorescence double-staining with anti-c-Myc antibodies (A) and anti-mannosidase II antibodies (B). Overexpression of c-Myc-tagged p58 and immunofluorescence staining of permeabilized (C) and nonpermeabilized (D) cells with anti-c-Myc antibodies.
Overexpression of the c-Myc-tagged p58 resulted in the accumulation of the protein in the intracellular membranes. No increase in punctate elements could be detected while the ER network was heavily stained, and the Golgi complex, identified by double-staining with anti-mannosidase II antibodies (not shown), appeared enlarged (Fig. 6, panel C). When non-permeabilized cells were stained with anti-c-Myc antibodies, plasma membrane patches could be visualized (panel D), indicating that overexpression of the c-Myc-tagged p58 led to its mislocalization and leakage from the intracellular membranes to the plasma membrane.
During the last several years p58 (Saraste et al., 1987; Saraste and Svensson, 1991; Lahtinen et al., 1992) and p53 (Schweitzer et al., 1988, 1990; renamed ERGIC-53, Schindler et al.(1993)), two membrane proteins identified in rat and human cells, respectively, have been widely used as markers for the intermediate compartment at the ER-Golgi interface. These proteins have very similar intracellular distributions and biochemical properties, but their structural and functional relationship has been unknown. The abundance of p58 in pancreatic microsomal membranes (Lahtinen et al., 1992) allowed us to purify the protein to homogeneity, obtain peptide sequences, and clone the cDNA. The sequence reveals that rat p58 and human ERGIC-53 (Schindler et al., 1993) are homologous proteins sharing 89% identity at the amino acid level. Furthermore, the observed 71% identity between the isolated partial X. laevis p58 sequence and the rat sequence indicates a high degree of evolutionary conservation of the protein among different species.
The cDNA sequence, together with biochemical data, suggest that p58 is synthesized as a 517-amino acid protein having a predicted and cleavable signal sequence and a single transmembrane domain close to the C terminus. This type I membrane topology was confirmed by using peptide antibodies generated against lumenal and cytoplasmic domains of p58. Although p58 has been suggested to contain immature N-glycans (Hendricks et al., 1991), no consensus sites for N-glycosylation were found.
The expression of c-Myc-tagged p58 in BHK cells resulted in a
distribution identical to the endogenous protein (Saraste and Svensson,
1991). A typical punctate, Golgi-concentrated pattern could be
visualized by immunofluorescence microscopy. In cells overexpressing
p58, the protein appeared to accumulate mostly in the Golgi complex and
could also clearly be detected in the ER, but no increase in vesicular
elements was observed. This is in contrast to the vesicular pattern and
suggested enlargement of the intermediate compartment observed in Vero
cells overexpressing ERGIC-53 (Schindler et al., 1993). In rat
pancreas the staining of p58 in actively secreting acinar cells is more
punctate and disperse, as compared to the reticular Golgi-staining
pattern of -cells (Lahtinen et al., 1992). It is thus
possible that the distribution of p58 at the ER-Golgi interface
correlates with the secretory status of the cell. In fibroblasts, where
the level of secretion is relatively low, the amount of cargo proteins
and/or cytosolic factors, e.g. coatomers, could be limiting in
cells overexpressing p58, and therefore the production of anterograde
and/or retrograde transport vesicles would not be enhanced. This could
result in the preferential accumulation of the protein in donor or
acceptor organelles, respectively.
In non-permeabilized cells patchy staining of the cell surface, sometimes concentrated close to the cell edges, could also be visualized. Whether these structures represent clathrin-coated pits or caveolae is presently unknown. The same surface staining pattern has also been described for ERGIC-53 in overexpressing COS cells (Kappeler et al., 1994). This is likely to be due to saturation of the intracellular components needed for the retention of p58, resulting in leakage of the protein to the plasma membrane. The cytoplasmic tails of p58 and ERGIC-53 contain a double-lysine motif (Nilsson et al., 1989; Jackson et al., 1990), which mediates retrieval of membrane proteins from the Golgi to the ER (Jackson et al., 1993; Townsley and Pelham, 1994; Gaynor et al., 1994). Overexpressed ERGIC-53 has been shown to be endocytosed from the plasma membrane (Kappeler et al., 1994). Surprisingly, the C-terminal amino acid sequence K-K/R-F/Y-F/Y needed for internalization was found to be related to the KKXX ER retrieval signal (Itin et al., 1995).
The
immunofluorescence staining pattern obtained with the peptide
antibodies made against the short cytoplasmic tail appeared to differ
from the staining seen with antibodies made against a lumenal sequence.
Anti-tail antibodies recognized the subpopulation of p58 residing in
the Golgi cisternae, indicating that the epitope is masked or not
accessible in the vesicular elements, and exposed only in the Golgi
complex. Knowing the distribution of p58 in the Golgi complex (Saraste et al., 1987), this is predicted to be the cis cisternae of the Golgi. Whether anti-tail antibodies can be used
as a specific cis-Golgi marker has to be resolved at the
electron microscopic level. The differential exposure of the
double-lysine ER retrieval signal might regulate the trafficking of p58
by at least two mechanisms. First, the accessibility of the
double-lysine to the retrieval machinery might correlate with different
homo-oligomeric forms of p58 (Lahtinen et al., 1992). For
example during the oligomeric assembly of the IgE receptor the
-chain is retained in the ER. Steric masking of the two lysines
when the assembly with the
-chain has been completed allows the
receptor complex to be transported from the ER (Letourneur et
al., 1995). Second, specific protein-protein interactions might be
responsible for the transport to the Golgi complex, and recycling back
to the ER. Some of the coatomer subunits interact directly with
double-lysine containing peptides in vitro (Cosson and
Letourneur, 1994). Recently, coatomer subunits have been shown to be
essential for the retrieval pathway (Letourneur et al., 1994).
The ectodomains of p58 and VIP36, an integral membrane protein
isolated from exocytic carrier vesicles in epithelial Madin-Darby
canine kidney cells (Fiedler et al., 1994), were found to be
related, sharing 30% identity and 53% similarity. Both p58 and VIP36
are non-glycosylated type I integral membrane proteins but localize to
different parts of the secretory pathway. VIP36 was recently shown to
be related to leguminous plant lectins (Fiedler et al., 1994).
Although the similarity at the primary amino acid level was quite low,
the secondary structure and critical amino acids involved in sugar
residue and metal binding were conserved. As a consequence of the
lectin properties and the cellular localization a possible function in
the sorting of glycolipids and GPI anchored proteins was proposed
(Fiedler et al., 1994). Based on the sequence homology between
VIP36 and ERGIC-53, Fiedler and Simons(1994) postulated a new family of
animal lectins operating at different stages along the secretory
pathway. It is worth mentioning that the critical three amino acids
needed for lectin activity pointed out in the ERGIC-53 sequence are
conserved both in the rat and frog proteins. The argument for a
possible lectin function was strengthened, when MR60, a human
mannose-binding lectin purified by affinity chromatography, was cloned
and found to be identical to ERGIC-53 differing only in one amino acid
residue (Pimpaneau et al., 1991; Arar et al., 1995).
MR60 was also shown to have structural similarities with galectins,
animal -galactoside binding lectins. Lectin-like affinity for
glycoproteins containing terminal mannoses might be needed for sorting
and concentrating proteins into transport vesicles in the ER (Mizuno
and Singer, 1993; Balch et al., 1994; Pelham, 1995). This
would be followed by delivery of the ligand in the the Golgi complex,
and the recycling of unoccupied receptor back to the ER. Alternatively,
p58 could have a function for retrieval of escaped, not fully matured,
or folded glycoproteins back to the ER.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U44129 [GenBank](rat) and U44130 [GenBank](X. laevis).