Identification of Potential Regulatory Elements for the Transport of Emp24p

Abstract

To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxylterminal region of Emp24p. Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed. The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p. Mutational analyses revealed that the two carboxylterminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport. The acceleration was sequence specific, and the terminal valine appeared to be more important. The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export. Hence the LV residues are a novel anterograde transport signal. The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal. On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step. It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down. Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi. Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

“…The C-terminal half of the protein is predicted to form a long helical structure extending across the transmembrane domain to the C-terminal cytosolic tail, which is implicated in the complex formation (58 -60). The cytosolic tail contains one or more of the known COPI/ COPII binding motifs including the di-phenylalanine motif, the di-lysine motif, and the C-terminal valine motif (49,60,61). A secondary structure prediction of Rrt6 suggests that these characteristics are well conserved in this protein.…”

“…Mutations of the last two amino acids (GL) modestly increased the amount of Rrt6 in the ER-enriched P13 fraction. So these residues might function as an ER export signal like the C-terminal valine motif in Emp24 (60,61).…”

Diacidic Motifs Influence the Export of Transmembrane Proteins from the Endoplasmic Reticulum in Plant Cells

“…The cytoplasmic sorting motifs are usually divided into two groups, the dihydrophobic and diacidic signals (reviewed in Barlowe, 2003), although a new group of dibasic signals was recently proposed (Giraudo and Maccioni, 2003). Dihydrophobic export signals generally contain two large hydrophobic or aromatic amino acid residues adjacent to one another and are usually found toward the C terminus of the protein (Fiedler and Rothman, 1997;Kappeler et al, 1997;Dominguez et al, 1998;Nakamura et al, 1998;Otte and Barlowe, 2002;Sato and Nakano, 2002). Diacidic motifs consist of two acidic amino acid residues, separated by any other amino acid, usually denoted DXE or Asp-X-Glu (Nishimura and Balch, 1997;Nishimura et al, 1999;Sevier et al, 2000;Ma et al, 2001;Votsmeier and Gallwitz, 2001), although DXD (Asp-X-Asp) motifs have also been shown to be functional (Malkus et al, 2002;Wang et al, 2004).…”

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

“…This view is supported by the recent observation that CFTR associates with CAL in the trans-Golgi network (42) and that the PDZ-interacting domain of pro-TGF-α is required for its efficient trafficking along the secretory pathway and for its expression at the plasma membrane (45). Moreover, the COOH-most terminal amino acids of several proteins are required for the efficient export of the proteins from the endoplasmic reticulum and/or the trafficking of these proteins to the plasma membrane (46,47). Third, the PDZ-interacting domain may be a apical sorting determinant that directs the trafficking of CFTR from the trans-Golgi network to the apical membrane.…”