Topological Regulation of TM4SF20

Transmembrane proteins must adopt proper topology to perform their functions. It was previously demonstrated that ceramide regulates TM4SF20 (transmembrane 4 L6 family 20) by altering the topology of the transmembrane protein, but the underlying mechanism remains obscure. This regulatory mechanism, denoted regulated alternative translocation (RAT), depends on a GXXXN motif present in the first transmembrane helix of TM4SF20. In the first part, using site-directed mutagenesis, I show that Asn-26 in the motif is crucial for RAT of TM4SF20, as it cannot be replaced even by Gln. In contrast, Gly-22 could be substituted by other small residues such as Ala and Ser without affecting RAT of TM4SF20. I further demonstrate that the GXXXN motif alone is insufficient to induce RAT because TM4SF4, a relative of TM4SF20 that also contains the motif in the first transmembrane helix, does not undergo RAT. Using TM4SF40-TM4SF20 chimeras, I determined that Pro-29 is also important for RAT of the protein: Replacing Pro-29 together with either Leu-25 or Val-17 of TM4SF20 with the corresponding residues of TM4SF4 abolished RAT of TM4SF20. Because Val-17, Gly-22, Leu-25, Asn-26, and Pro-29 are predicted to reside along the same surface of the transmembrane helix, our results suggest that interactions with other proteins mediated by this surface during translocation may be critical for RAT of TM4SF20. In the second part, I revealed the mechanism behind RAT of TM4SF20. TM4SF20 is synthesized in the endoplasmic reticulum (ER) with a cytosolic C terminus and a luminal loop before the last transmembrane helix where N132, N148, and N163 are glycosylated. In the absence of ceramide, the sequence surrounding glycosylated N163 but not N132 is retrotranslocated from lumen to cytosol independent of ER-associated degradation. Accompanying this retrotranslocation, the C terminus of the protein is relocated from cytosol to lumen. Ceramide delays the retrotranslocation process, causing accumulation of the protein that is originally synthesized. These findings suggest that N-linked glycans, although synthesized in the lumens, may be exposed to cytosol through retrotranslocation, a reaction that may play a crucial role in topological regulation of transmembrane proteins.

Author dc:creator

• Wang, Jingcheng

Contributors dc:contributor

• Liu, Yi
• Ye, Jin
• Chen, Zhijian J.
• DeBose-Boyd, Russell A.
• Kittler, Ralf

• Glycoproteins
• Glycosylation
• Membrane Proteins
• Endoplasmic Reticulum
• Polysaccharides

Language dc:language

en

Identifier

1535537230

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10702