Presentation to Fulfill the paper study work By

Birendra Kumar SinghI.D. 22131690

Melanin pigments are synthesized and stored in cell type specific, membrane-bound compartments termed melanosomes.

Within melanosomes, melanogenic enzymes (e.g. tyrosinase) are integrally associated to membranes, whereas pigments are deposited in the lumen along a characteristic array of fibrous striations.

Although it is well known that the formation of these striations precedes melanin synthesis and occurs in an early stage of melanosome maturation, the nature of the striations and the molecular events that result in their formation have remained elusive for decades.

Recent evidence demonstrates that proteolytic processing of a melanocyte specifc protein in a post- Golgi compartment is a key event in melanosome ontogenesis because it releases a polypeptide that can generate the characteristic fibrous striations.

Stages I and II(also referred to as premelanosomes) being the most immature forms.

Melanosomes at stages II–IV can be defined as those containing fibrous striations and no pigment (stage II).

Melanin deposited along the striations (stage III)

Filled with melanin (stage IV).

Stage I melanosomes have been defined less precisely, leading to different models of their possible origin

There are two views for the possible origin of stage I melanosome.

One view is that stage I melanosomes arise directly from the endoplasmic reticulum(ER), a notion supported by the recent detection of ER-resident proteins in a purified pre-melanosome preparation.

Another view is that premelanosomes arise from compartments of the late secretory (post-Golgi) and/or endocytic pathways

In either case, there is compelling evidence that the melanogenic enzymes are trafficked through the Golgi before reaching premelanosomes (presumably at stage II), and that there is an overlap between the biogenesis of melanosomes and lysosomes.

One approach used to understand the origin of premelanosomes is the study of a protein enriched in these compartments, Pmel17.

Pmel17, also known as ME20, gp100, RPE1 and silver locus protien, was identified as a specific marker of melanosomes and subsquently, as the product of the muriene silver pigmentation gene.

Observations suggest that Pmel17 could be processed by proteolysis and localize to the melanosomal lumen.

The primary structure of Pmel17 predicts a type I integral membrane protein with a large lumenal domain, a single transmembrane domain and a short cytoplasmic tail (Figure 2).

In agreement with this prediction, most antibodies raised to Pmel17 recognize a bona fide integral membrane protein.

However, a secreted fragment of Pmel17was isolated from a melanoma cell culture, and antibodies raised to a protein mixture extracted from the melanosomal lumen were able to recognize Pmel17 .

These observations suggest that Pmel17 could be processed by proteolysis and localize to the melanosomal lumen.

Both Interpretations have been corroborated and found to reflect important events in melanosome biogenesis.

Using immunoelectron microscopy on ultra-thin cryosections, Raposo et al. examined the distribution of Pmel17 in MNT-1, a highly pigmented human melanoma cell line that produces morphologically normal premelanosomes and melanosomes.

Using antibodies to the lumenal domain of the protein, they observed profuse labeling of the striations of stage II melanosomes and, to a lesser extent, of a compartment named the ‘coated endosome’ (owing to the presence of large clathrin lattices on the cytoplasmic side) and other organelles such as the Golgi.

The coated endosome is also found in nonpigmented cells and is an endosomal precursor of multivesicular bodies (MVBs).

Similar experiments using antibodies to the cytoplasmic tail of Pmel17 resulted in labeling of the coate endosome and Golgi but not of the premelanosome striations, suggesting that the cytoplasmic tail is either buried or removed upon association of Pmel17 to the striations.

Pmel17 not only localizes to the striations but it is sufficient to generate them. Upon overexpression of Pmel17 in human HeLa cells, fibrous striations were formed within MVBs that were morphologically similar to those of premelanosomes in pigmented cells.

As in MNT-1 cells, the striations were densely labeled with antibodies to the Pmel17 lumenal domain but not with antibodies to the cytoplasmic tail.

The post-translational processing of Pmel17 was examined in detail by Berson and co-workers (Figure 2).

In pulse–chase analysis, the first Pmel17 species detected (P1) corresponded to the protein modified with high-mannose oligosaccharides, a modification that normally occurs in the ER.

A second species (P2) and, slightly later, two fragments (Ma and Mb) accumulated upon disappearance of P1 during the chase period.

Unlike P1, the P2, Ma and Mb species were all resistant to digestion by endoglycosidase H, which is a hallmark of the trafficking of glycoproteins through the Golgi. Similar kinetics of accumulation of Ma and Mb, and the fact that they could associate with each other through a disulfide bond, indicated that they might arise from cleavage of Pmel17 by an endoprotease

The search for the endoprotease responsible for Pmel17 processing benefitted from the earlier characterization of a polypeptide secreted from melanoma cells, which was identified as Pmel17 residues 25–467.

This polypeptide is probably identical to Ma, as suggested by their similar electrophoretic mobilities and the fact that Ma was also detected in the culture medium of growing melanoma and transfected HeLa cells.

Three lines of experimental evidence from Berson et al. suggest that furin or a related family member is required for normal Pmel17 processing.

(i) Mutation of the lysine–arginine pair to a glutamine pair (KR ! QQ) precluded processing of Pmel17 into Ma and Mb fragments in transfected HeLa cells.

(II) No Mb (and presumably no Ma) waproduced upon expression of wild-type or KR ! QQ versions of Pmel17 in human LoVo tumor cells, which are deficient in furin and other proprotein convertases. Coexpression of furin in these cellsre stored the generation of Mb fragment from wild-type Pmel17 but not from the KR ! QQ mutant.

(III) Expression in MNT-1 cells of a1-antitrypsin Portland, a version of a1-antitrypsin engineered to inhibit furin selectively, dramatically inhibited the processing of endogenous Pmel17 into Ma and Mb, whereas the original a1-antitrypsin had no effect.

Moreover, the groups of Marks and Raposo have demonstrated that such proteolytic processing of Pmel17 is necessary for the formation of the fibrous striations

The findings described in this article indicate that the release of a fragment (Ma) from the lumenal domain of Pmel17, mediated by a proprotein convertase, is a key event in the formation of melanosomal striations.

Besides improving our understanding of the formation of the fibrous striations of melanosomes, the observations made by the groups of Marks and Raposo have provided two arguments against the hypothesis that stage I melanosomes arise directly from the ER.

(i) If stage I melanosomes were defined as organelles enriched in Pmel17 and lacking striations, they would correspond to the coated endosomes, which clearly are components of the endocytic pathway.

The fact that processed forms of human Pmel17 are largely resistant to endoglycosidase H implies that the majority of the protein is trafficked through the Golgi before reaching premelanosomes (modification of Pmel17 with sialic acid, which typically occurs in the Golgi, has also been demonstrated [17]). Furthermore, the crucial cleavage of Pmel17 to release the Ma fragment probably occurs at the trans-Golgi network or in endosomes, because these are the normal sites of action of furin and related enzymes.

Nevertheless, further work is required to understand the ontogenesis of melanosomes fully.

Factors other than Pmel17 are probably required to generate a stage II melanosome as a distinct organelle because the striations formed in HeLa cells upon Pmel17 overexpression seem to accumulate within typical MVBs