Why are glycoproteins destined for secretion

Yeast cytosol was prepared from the XA strain as described before Wuestehube and Schekman, Subcellular fractionation was performed as described before Powers and Barlowe, Cells were grown to an early log phase and were converted to spheroplasts by Zymolyase treatment.

Thriteen fractions of 1 ml each were taken from the top to the bottom. To characterize the membrane association of Emp46p and Emp47p, yeast lysate was isolated and treated with agents as follows. Spheroplasts were prepared as indicated above and resuspended in buffer Samples were incubated on ice for 30 min followed by centrifugation at 70, rpm model TLA The secretion of glycoproteins into the medium was measured as described earlier Yahara et al.

Intracellular and extracellular fractions were separated by centrifugation and the supernatant fractions were added to the equal volume of mM Tris-Cl, pH 7. Specifically to visualize labeled glycoproteins, the samples were incubated with Concanavalin A conA -Sepharose Amersham Pharmacia Biotech. The beads were washed twice with a buffer 50 mM Tris-Cl, pH 7.

Emp47p is a nonglycosylated type-I membrane protein with a dilysine signal in its cytoplasmic tail, and it has previously been shown to recycle between the Golgi complex and the ER Schroder et al. Like Emp47p and its mammalian homolog ERGIC , YLRw protein has a single potential transmembrane domain at its C terminus as revealed by the hydrophobicity plot Kyte and Doolittle, , and a residue segment in the N-terminal region that shares homology with the carbohydrate-recognition domain of several leguminous plant lectins Figure 1 , B and C.

Because the high degree of identity suggests that the homolog might serve a very similar function in the cell, we decided to study both Emp47p and YLRw protein. A YLRwp is homologous to Emp47p. B Hydrophobicity profiles calculated according to Kyte and Doolittle with a window size of 6.

C The schematic diagram of the polypeptide chain and the type I topology of Emp46p indicates the positions of its functional domains. A polyclonal antibody was raised against the N-terminal region of YLRw protein amino acid positions 1— expressed from E. The protein sequence upstream of the N terminus of the mature polypeptide is hydrophobic in nature and conforms with the rules for cleavable signal peptides von Heijne, The presence of a signal peptide and the C-terminal transmembrane domain suggests that the protein assumes a type-I transmembrane protein.

These features are similar to those previously reported for Emp47p Schroder et al. To explore the function of EMP46 and EMP47 , null mutants were generated, and isogenic single and double mutants were analyzed. This defect is completely rescued by the addition of the osmotic stabilizer sorbitol 1 M to the growth medium Figure 2 A. F Emp46p and Emp47p do not depend on each other for their expression. Kar2p is shown as a control. The high level of amino acid sequence similarity between Emp46p and Emp47p suggests that they might be redundant proteins with similar functions in the cell.

Cells deleted for EMP46 , EMP47 , or both were also analyzed for their growth properties in the presence of high concentrations of divalent cations. Because of failure to detect endogenous Emp46p with anti-Emp46p antibodies used in N-terminal peptide sequence, a 3HA tag was after the signal peptidase cleavage site at amino acid position 3 of Emp46p in a single-copy 3HA-Emp46p expression plasmid.

To confirm that Emp46p is indeed an integral membrane protein, we examined its fractionation behavior. The fractionation profile for Emp46p was the same as for Emp47p, as they could only be solubilized by detergent and not by carbonate treatment Figure 3 A.

A Emp46p is an integral membrane protein. Fractions were probed by immunoblotting. All data are from a single gradient, plotted in two parts for clarity. Relative levels of Emp46p, Kex2p, Sec61p, Pho8p, and Emp47p as determined by densitometry of the immunoblots are shown in C. Twenty percent of a total lysate is shown as T. We next examined the subcellular localization of Emp46p by resolution of membrane organelles on sucrose gradients Figure 3 , B and C. Emp46p sedimented in two peaks, one that coincided with the cis -Golgi marker Emp47p, and the second smaller peak cosedimenting with the ER marker Sec61p.

Emp47p has previously been shown to recycle between the Golgi and the ER, which requires its C-terminal dilysine signal Schroder et al. The punctate structures disappeared and a ring around the nucleus and some fluorescence in the periphery of the cell, which are typical for yeast ER, became visible.

This relocalization is reversible. The dynamics of intracellular distribution of GFP-Emp46p in sec cells. The same cells are shown. Images were visualized by confocal laser microscopy. The left panels show the corresponding Nomarski images.

The same effect was observed with an independent experimental approach. The majority of Emp46p that was present in the middle of the gradient i. We conclude that like Emp47p, Emp46p localize to the early secretory pathway and recycles between the Golgi complex and the ER. Emp47p has previously shown to contain a functional dilysine ER-recycling signal, and mutations in this signal led to a rapid transport of the protein to the vacuole Schroder et al.

To see whether the C-terminal dilysine signal of Emp46p Figure 6 A also functions as a retrieval signal, this motif was disrupted by replacing double lysine residues with serine or arginine residues. The same effect could be observed by sucrose density gradients.

Figure 7 shows the distribution of Emp46p dilysine mutants on sucrose gradients; they were virtually absent in fractions containing Sec61p Figure 7 , fractions 8— We could not detect the signal in vacuole fractions either, probably because the PEP4 gene, which is responsible for major vacuolar proteolysis, was not knocked out in the cells used in the fractionation experiments.

A The C-terminal region of Emp46p and other traffic lectins. ER-targeting dilysine signals are boldfaced, and the tyrosine-containing motifs are underlined. B Influence of the C-terminal region of Emp46p on its intracellular steady-state localization. The left panels show the GFP patterns, and the right panels show the corresponding Nomarski images. A whole-cell lysate from cells expressing 3HA-Emp46p with a wild-type or mutated dilysine motif was fractionated on a sucrose density gradient identical to that shown in Figure 3.

Fractions were analyzed by immunoblotting. Quantitation was by densitometric scanning of immunoblots. A whole-cell lysate from cells expressing 3HA-Emp46p with a wild-type or mutated tyrosine-containing motif was fractionated on a sucrose density gradient and analyzed as above.

In yeast Emp46p and Emp47p, the diphenylalanine motif is replaced by two leucines Figure 6 A. A previous report showed that the exchange of the two C-terminal leucines of Emp47p with alanines did not change the wild-type distribution Schroder et al.

Furthermore, there was a clear difference in the fractionation properties of mutant Emp46p compared with Emp46p with original cytoplasmic tail Figure 7 A. These results demonstrate that the most C-terminal two leucines contribute to efficient ER exit of Emp46p. Such a tyrosine-based motif has been identified to be involved in recognition by adapter complexes Mallabiabarrena et al.

We mutated this region to alanine and compared the localization of wild-type and mutant Emp46p fused to GFP. In contrast to wild-type Emp46p, a single substitution at any of the conserved residues within the tyrosine-containing motif exhibited a drastic decrease of Golgi localization Figure 6 B, b-d , with a fluorescence pattern typical of the ER, indicating that the first tyrosine and the last hydrophobic residue are required for normal Emp46p exit from the ER.

Alanine substitution of the tyrosine-containing motif caused a moderate shift of mutant Emp46p to the ER membrane fractions Figure 7 B , which also represents the contribution of the C-terminal tyrosine-containing motif to Emp46p localization.

It should be noted that the punctate structures observed with GFP-Emp46p tyrosine-motif mutants are not the result of mislocalization to an endosomal compartment. The late endosomal syntaxin, Pep12p, clearly does not copeak with Emp46p tyrosine-motif mutants Figure 7. Under the reconstituted in vitro budding reaction, ER-derived COPII vesicles incorporated wild-type Emp46p at a level comparable with other characterized cargo proteins, such as Sec22p Barlowe et al.

To test specific packaging of the C-terminal mutants of Emp46p, microsomes were prepared from these strains and the relative packaging efficiencies were determined. The tyrosine-containing motif mutant AYMA was packaged to a significantly lesser extent 1. These results show a direct defect in anterograde transport of C-terminal mutants.

A In vitro budding reactions with microsomes prepared from cells expressing both 3HA-Emp46p and 2myc-Emp47p. Sec61p as negative control and Sec22p as positive control were detected with polyclonal antibodies. B In vitro budding reactions with microsomes prepared from strains expressing C-terminal mutants of Emp46p. The same protocol as in A was used except that the reaction time was 15 min.

The fusion protein was purified from E. The beads were then incubated with yeast lysate. As with the Emp47p tail, the binding was dependent on the two conserved lysine residues because binding was completely abolished when these two lysines were substituted for serines Figure 9. Both Ret1p and Sec21p were also detected in proteins bound to the GST tail protein, with the most C-terminal two leucines replaced by alanines.

In contrast, less Ret1p and Sec21p were observed in proteins bound to the GST tail with mutated tyrosine-containing motifs. This binding requires both of the conserved tyrosine and hydrophobic residues, as replacement of both tyrosine and phenylalanine to alanines synergistically affected the COPI binding. These results suggest that in addition to the dilysine motif, the C-terminal tyrosine-containing motif of Emp46p contributes to COPI binding.

Under the same experimental conditions, C-terminal tails were also tested for their possible binding of COPII by looking at the binding of Sec23p. Sec23p binding was seen to the GST tail of Emp46p and Emp47p, but also to dilysine mutants in which the two lysines were replaced with serines. However, no decrease of Sec23p binding was observed when the C-terminal two leucines were mutated to double alanine; this was unexpected because this mutant showed a greater dependency on this motif for the ER exit Figures 6 B, e, 8B, and 9A.

Instead, we observed the influence of the tyrosine-containing motif in modulating COPII binding because changing tyrosine, phenylalanine, or both completely abolished binding to Sec23p Figure 9. These were examined in greater detail by testing whether those motifs were necessary for the recovery of Emp46p in detergent-soluble prebudding complexes Figure Recovery in this prebudding complex is specific as ER resident proteins are not included in this intermediate. In contrast, when the tyrosine-containing motif or C-terminal double leucine was mutated to alanine, Emp46p was absent from the prebudding complex, whereas control Sec22p was always present in the complex.

Therefore, we conclude from these experiments that the anterograde transport of Emp46p is due to a combined action of the tyrosine-containing motif and the C-terminal two leucines.

The role of the C-terminal tail of Emp46p in recruitment into the prebudding complex. Subsequently, digitonin-soluble prebudding complexes were recovered on glutathione beads. Emp46p or Sec22p in the prebudding complex was detected by immunoblotting.

T represents 0. Each construct is denoted as described in the legend to Figure 6 B. Fawcett, D. Saunders, Philadelphia. Fine, R. Flynn, G. Freedman, R. Freedman and H. Hawkins, eds. Fujiwara, T. Fuller, S. Gallagher, P. Galteau, M. Ganem, D. Geetha-Habib, M. Gerace, L. Gething, M. Goud, B. Griffiths, G.

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Meyer, D. Meyer, J. C, Bergmann, C. Mundy, D. Munro, S. Navarro, D. Newman, A. Nilsson, T. The various membranes involved, though interrelated, differ in structure and function. The endomembrane system plays a very important role in moving materials around the cell, notably proteins and membranes the latter is called membrane trafficking.

For example, while many proteins are made on ribosomes that are free in the cytoplasm and remain in the cytoplasm, other proteins are made on ribosomes bound to the rough endoplasmic reticulum RER.

The latter proteins are inserted into the lumen of the RER, carbohydrates are added to them to produce glycoproteins, and they are then moved to cis face of the Golgi apparatus in transport vesicles that bud from the ER membrane.

Within the Golgi, the protein may be modified further and then be dispatched from the trans face in a new transport vesicle. These vesicles move through the cytoplasm to their final desinations using the cytoskeleton.

We can think of the system as analogous to a series of switching yards and train tracks, where materials are sorted with respect to their destinations at the switching yards and sent to those destinations along specific tracks in the cytoskeleton. Proteins destined for secretion are made on ribosomes bound to the RER. The proteins move through the endomembrane system and are dispatched from the trans face of the Golgi apparatus in transport vesicles that move through the cytoplasm and then fuse with the plasma membrane releasing the protein to the outside of the cell.

Examples of secretory proteins are collagen, insulin, and digestive enzymes of the stomach and intestine. In a similar way, proteins destined for a particular cell organelle move to the organelle in transport vesicles that deposit their contents in the organelle by membrane fusion.

Like secretory proteins and some other proteins, proteins destined for lysosomes are made on ribosomes bound to the RER and move through the endomembrane system. In this case the lysosomal protein-containing vesicle that buds from the trans face of the Golgi apparatus is the lysosome itself.

The figure below illustrates at a glance the structures that are common to both animal and plant cells, as well as the structures that are unique to each. Structures that are common to both plant and animal cells are labeled between the cells; structures that are unique to plants are labeled on the left of the cells and those unique to animals are labeled on the right.

Chloroplasts are plant cell organelles that contain chlorophyll and the enzymes required for photosynthesis, the light-dependent synthesis of carbohydrates from carbon dioxide CO2 and water H2O.

Oxygen O2 is a product of the photosynthesis process, and is released into the atmosphere. Chloroplasts are large organelles bounded by a double membrane and containing DNA. Unlike the mitochondrial double membrane, the inner membrane is not folded.

Distinctly separate from the double membrane is an internal membrane system consisting of flattened sacs and called thylakoids. The space between the thylakoid and the outer membranes is called the stroma. The stroma contains the chloroplast DNA as well as components of the protein synthesizing machinery specific for the chloroplast, namely the ribosomes, tRNAs, and specific proteins and enzymes.

Most of the components of photosynthesis are located in the thylakoids. The thylakoid membranes are organized into stacks called grana. The interior of the thylakoid is the lumen.