This work completes the initial characterization of Emp46p with significant homology to Emp47p. Like Emp47p, Emp46p is an integral membrane protein and is predicted to have a large lumenal domain with a shorter C-terminal cytoplasmic segment. The lumenal domain of Emp46p shows significant homology to its mammalian homolog ERGIC-53, which has been shown to have a lectin activity. The emp47
deletion in our genetic background has clear phenotypes, irrespective of an earlier report (Schroder et al., 1995
). Strains carrying the emp47
Δ null mutation showed thermosensitivity for growth. The emp47
Δ mutation also influenced the Ca2+
sensitivity of the cells. This may be due to two fundamentally different mechanisms. Either Emp46/47p operate the factors that are involved in calcium homeostasis or the proper binding and release of substrates to the lectin domain is disturbed. We consider the latter possibility unlikely because the double mutant also showed sensitivity to elevated levels of Ca2+
. Further experiments on calcium metabolism in the emp46
Δ and emp47
Δ strains are underway.
The high degree of similarity between Emp47p and Emp46p suggested that the latter might fulfill a very similar function. Complementation of both temperature and Ca2+ sensitivities were observed in experiments where Emp46p was expressed from a high-copy vector in a strain lacking Emp47p. These results suggested that the Emp46p function is partially redundant with Emp47p.
By analytical fractionation, the majority of Emp46p was found in compartments whose density was similar to that of Emp47p, which was already proven to locate in the Golgi. Localization studies by GFP labeling show that although Emp46p resided largely in the Golgi at steady state, this protein constitutively recycles between the Golgi and the ER. Our in vivo analyses and in vitro assays indicate that the C-terminal tail region imparts steady-state localization to Emp46p. Previous reports indicated that the dilysine motif contained on the C-terminal tail of Emp47p is important for its localization and can mediate association with the COPI coat complex (Schroder et al., 1995
; Schroder-Kohne et al., 1998
). Our findings demonstrate that, like Emp47p, the Emp46p tail sequence possesses a dilysine motif that assists in binding subunits of the COPI coat. Lysine-to-serine mutations within this motif resulted in redistribution of Emp46p to the vacuole. This result can be readily explained by the importance of these residues for recruitment of COPI coat and Golgi-to-ER retrieval. The COPI coat was shown to be recruited in vitro. The GST tail of Emp46p showed COPI binding in what appeared to be a dilysine-restrictive manner, and in comparable amounts with the positive control, Emp47p, which has been shown to display a functional dilysine motif (Schroder-Kohne et al., 1998
). The clear reduction of COPI binding was observed when the lysines within the motif were mutated to serines.
A previous report described that the steady-state distribution of Emp47p with the C-terminal two leucines replaced by alanines showed a punctate pattern indistinguishable from the wild type (Schroder et al., 1995
). Our findings are not entirely consistent with this study; however, more precise analyses were performed. Experiments with GFP labeling revealed that the C-terminal two leucines are strictly required for the ER exit. Unexpectedly, however, our in vitro binding experiments revealed that the binding of the COPII component Sec23p to the C-terminal region of Emp46p was independent of the two C-terminal leucines. In contrast, the mutation of the two leucines to alanines decreased the ability of Emp46p to be recovered in detergent-soluble prebudding complexes. We conclude that the C-terminal double leucine is a part of an ER exit determinant, which is required for the cargo concentration at the prebudding site. These results raise the possibility that the multiple signals may be required in Emp46p for efficient export.
Interestingly, we found that a sequence similar to the previously identified tyrosine-containing motif that mediates adaptor and COPI binding (Mallabiabarrena et al., 1995
; Ohno et al., 1998
) is located at the border of the transmembrane domain of this protein family, which contributes steady-state localization of Emp46p. Cells expressing the GFP-Emp46p protein with a mutated tyrosine-containing motif had clear ER staining, suggesting that this motif affects the ER exit of Emp46p rather than COPI-mediated retrieval. This is reminiscent of the recent observations that in addition to the DXE motif of the VSV-G protein, an upstream tyrosine-containing motif is also involved in efficient ER export (Sevier et al., 2000
; Aridor et al., 2001
). The decreased rate of ER exit of Emp46p with mutations in the tyrosine-containing motif is not likely to be due to the quality control in the ER. Our in vitro binding experiments with purified GST tail fusion constructs demonstrated that the change of any conserved feature within the tyrosine-containing motif completely abolished the Sec23p binding. Substitution of the conserved residues with alanines also decreased the binding of COPI components, although not as efficiently as the COPII binding. The observation that the Emp46p protein strongly binds both COPI and COPII predicts a competitive recruitment of COPI and II for this family member. It is not known how the same C-terminal region can participate in diverse transport steps and how it interacts with different coats at each step. It is conceivable that the dilysine motif at the C terminus of Emp46p is presented in an optimal way; for instance, this part is masked at the level of the ER and only accessible to COPI components when the protein arrives in the Golgi. Further studies will be necessary to precisely determine the similarities and the differences between the C-terminal motifs recognized by COPII and COPI coats.
It should be noted that only the cytosolic fraction of Sec23p binds specifically to GST tail fusion proteins, and this interaction is dependent on the presence of conserved tyrosine and a hydrophobic residue within the motif. Indeed, we could not detect any interaction between purified COPII components, such as Sec23/24p or Sar1p, and GST tail protein comprising the C-terminal domain of the Emp46p (our unpublished observation). It may imply the existence of an adaptor-like protein in between Emp46p and COPII components.
Our results demonstrate the ability of Emp46p and Emp47p to influence the secretion efficiency of certain glycoproteins, suggesting that a model for these proteins serves as transport receptor for some glycoproteins. It seems unlikely that Emp46p and Emp47p act as chaperones for glycoproteins in the Golgi, a role previously proposed for Emp47p (Schroder et al., 1995
), because the steady-state localization of Emp46p to the Golgi is not required for its function. The secretion of Emp47p-dependent glycoproteins is minimally affected by the deletion of Emp46p, but the additive secretion defects of the emp46
Δ double deletions to some glycoproteins were also observed, suggesting that Emp46p and Emp47p share similar functional properties that are partially redundant or even substitute for each other. Several glycoproteins secreted from the emp47
Δ strain were underglycosylated, which raises the possibility that glycoproteins secreted from emp47
Δ cells have bypassed some Golgi glycosyltransferase activities and the underglycosylation is due to defects in mannose-chain elongation. A probable explanation would be that the activity of Emp47p may regulate the compartmental distribution of glycosyltransferases, which are involved in mannose elongation and addition within the Golgi.
Importantly, the deletion of EMP46 or EMP47 makes cells flocculate in the liquid culture (our unpublished observation). In addition, there is a synergistic effect caused by their double deletion, namely, a stronger tendency to flocculate in the liquid medium than either single mutant, providing further implication that Emp46p and Emp47p may participate in cell wall glycoprotein transport. This possibility is also consistent with the fact that sorbitol, an osmotic stabilizer, rescues the growth defect of the emp47 Δ strain at high temperature, although how this occurs is not clear.
In conclusion, we have shown that the Emp46p cycling between the Golgi apparatus and the ER requires both the C-terminal ER exit determinant and the ER retrieval signal that interacts with the COPII component Sec23p and COPI components, respectively. The presence of the ER exit determinant in Emp46p, which has lectin-like properties, provides further evidence that Emp46p may function as a sorting receptor for glycoproteins in the early secretory pathway. Indeed, our results demonstrate that functional Emp46p and Emp47p are required to increase the secretion efficiency of several glycoproteins. Further development of both in vivo and in vitro transport assays from the ER to the Golgi should help us to investigate the proposed functions of Emp46p and Emp47p in more detail.