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The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol.

Mosse CA, Meadows L, Luckey CJ, Kittlesen DJ, Huczko EL, Slingluff CL, Shabanowitz J, Hunt DF, Engelhard VH - J. Exp. Med. (1998)

Bottom Line: Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum.Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent.We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and the Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22904, USA.

ABSTRACT
Formation of major histocompatibility complex class I-associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201-associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

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Deglycosylation of membrane tyrosinase produces a band  which comigrates or nearly comigrates with the tyrosinase species found  in the cytosol. Lane 1 contains the membrane fraction from 5 × 105 cells  mock treated with PNGase F. Lanes 2–5 contain equivalent DM93 membrane fractions treated with 1.5 mU PNGase for 2, 4, 8 and 12 h, respectively. Lane 6 contains the cytosolic fraction of 5 × 105 DM93 cells  treated with lactacystin for 5 h.
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Figure 10: Deglycosylation of membrane tyrosinase produces a band which comigrates or nearly comigrates with the tyrosinase species found in the cytosol. Lane 1 contains the membrane fraction from 5 × 105 cells mock treated with PNGase F. Lanes 2–5 contain equivalent DM93 membrane fractions treated with 1.5 mU PNGase for 2, 4, 8 and 12 h, respectively. Lane 6 contains the cytosolic fraction of 5 × 105 DM93 cells treated with lactacystin for 5 h.

Mentions: The results obtained thus far suggested that initial translation of tyrosinase in the ER was followed by the export of the full-length protein or a fragment to the cytosol for processing by the proteasome. To investigate the nature of the tyrosinase species exported from the ER to the cytosol, we used lactacystin to inhibit proteasome activity in DM93, a tyrosinase-expressing melanoma, and NA8 Mel, a tyrosinase-negative melanoma. After 5 h the cells were divided into cytosolic and membrane fractions, separated by SDS-PAGE, and immunoblotted for tyrosinase (Fig. 9). In untreated DM93 cells, we observed a strong doublet of 70–75 kD that was located exclusively in the membrane fraction. This doublet represents two major glycosylated species of tyrosinase, and correlate well with those previously identified by others (61, 69). In DM93 cells that had been treated with lactacystin, three additional bands were observed at 56–58 kD, similar in size to the unglycosylated full-length tyrosinase. The band of lowest mobility among this group was located exclusively in the membrane fraction, whereas the other two were found only in the cytosol. To further identify the potential nature of these lower molecular weight bands, the membrane fraction from DM93 cells was treated with PNGase F to deglycosylate asparagine-linked glycosylations (Fig. 10). Human tyrosinase contains seven potential glycosylation sites (70, 71) yet it appears that only four or five sites are used in DM93 cells. Although deglycosylation was not complete after 12 h, the fastest migrating species comigrated with the cytosolic 57-kD band, which appears after lactacystin treatment of DM93 cells. One additional species with an apparent molecular weight of 85-kD was found in the cytosol of untreated cells, but increased in intensity upon lactacystin treatment. The size of this band is too high to be attributed to glycosylation, but is consistent with the attachment of four ubiquitin chains to the unglycosylated full-length tyrosinase polypeptide chain. After long exposure times, two additional low intensity bands of 61 and 65 kD were also found in the cytosol. None of these bands were observed in extracts of NA8 Mel (Fig. 9), whereas a similar pattern was observed in NA8 Mel + Tyr cells (data not shown), indicating that all of the species are derived from tyrosinase. These results are consistent with the hypothesis that the export of a full-length or almost full-length tyrosinase from the ER for degradation by the proteasome provides source material for the production of the YMDGTMSQV peptide epitope.


The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol.

Mosse CA, Meadows L, Luckey CJ, Kittlesen DJ, Huczko EL, Slingluff CL, Shabanowitz J, Hunt DF, Engelhard VH - J. Exp. Med. (1998)

Deglycosylation of membrane tyrosinase produces a band  which comigrates or nearly comigrates with the tyrosinase species found  in the cytosol. Lane 1 contains the membrane fraction from 5 × 105 cells  mock treated with PNGase F. Lanes 2–5 contain equivalent DM93 membrane fractions treated with 1.5 mU PNGase for 2, 4, 8 and 12 h, respectively. Lane 6 contains the cytosolic fraction of 5 × 105 DM93 cells  treated with lactacystin for 5 h.
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Related In: Results  -  Collection

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Figure 10: Deglycosylation of membrane tyrosinase produces a band which comigrates or nearly comigrates with the tyrosinase species found in the cytosol. Lane 1 contains the membrane fraction from 5 × 105 cells mock treated with PNGase F. Lanes 2–5 contain equivalent DM93 membrane fractions treated with 1.5 mU PNGase for 2, 4, 8 and 12 h, respectively. Lane 6 contains the cytosolic fraction of 5 × 105 DM93 cells treated with lactacystin for 5 h.
Mentions: The results obtained thus far suggested that initial translation of tyrosinase in the ER was followed by the export of the full-length protein or a fragment to the cytosol for processing by the proteasome. To investigate the nature of the tyrosinase species exported from the ER to the cytosol, we used lactacystin to inhibit proteasome activity in DM93, a tyrosinase-expressing melanoma, and NA8 Mel, a tyrosinase-negative melanoma. After 5 h the cells were divided into cytosolic and membrane fractions, separated by SDS-PAGE, and immunoblotted for tyrosinase (Fig. 9). In untreated DM93 cells, we observed a strong doublet of 70–75 kD that was located exclusively in the membrane fraction. This doublet represents two major glycosylated species of tyrosinase, and correlate well with those previously identified by others (61, 69). In DM93 cells that had been treated with lactacystin, three additional bands were observed at 56–58 kD, similar in size to the unglycosylated full-length tyrosinase. The band of lowest mobility among this group was located exclusively in the membrane fraction, whereas the other two were found only in the cytosol. To further identify the potential nature of these lower molecular weight bands, the membrane fraction from DM93 cells was treated with PNGase F to deglycosylate asparagine-linked glycosylations (Fig. 10). Human tyrosinase contains seven potential glycosylation sites (70, 71) yet it appears that only four or five sites are used in DM93 cells. Although deglycosylation was not complete after 12 h, the fastest migrating species comigrated with the cytosolic 57-kD band, which appears after lactacystin treatment of DM93 cells. One additional species with an apparent molecular weight of 85-kD was found in the cytosol of untreated cells, but increased in intensity upon lactacystin treatment. The size of this band is too high to be attributed to glycosylation, but is consistent with the attachment of four ubiquitin chains to the unglycosylated full-length tyrosinase polypeptide chain. After long exposure times, two additional low intensity bands of 61 and 65 kD were also found in the cytosol. None of these bands were observed in extracts of NA8 Mel (Fig. 9), whereas a similar pattern was observed in NA8 Mel + Tyr cells (data not shown), indicating that all of the species are derived from tyrosinase. These results are consistent with the hypothesis that the export of a full-length or almost full-length tyrosinase from the ER for degradation by the proteasome provides source material for the production of the YMDGTMSQV peptide epitope.

Bottom Line: Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum.Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent.We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and the Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22904, USA.

ABSTRACT
Formation of major histocompatibility complex class I-associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201-associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.

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