Distinct Roles for the Cytoplasmic Tail Sequences of Emp24p and Erv25p in Transport between the Endoplasmic Reticulum and Golgi Complex

Abstract

Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K d ϳ100 M), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER.The secretory pathway in eukaryotic cells consists of a series of membrane-bound compartments that modify, sort and transport secretory cargo. Transport through this pathway depends on coat protein complexes that form vesicles and select specific cargo molecules for incorporation into vesicles. Current models suggest that transport between organelles is bi-directional, such that organellar constituents are recycled as secretory cargo advances. With regard to transport through the early secretory pathway, coat protein complex II (COPII) 1 catalyzes anterograde transport between the ER and Golgi whereas coat protein complex I (COPI) acts in retrograde traffic between these compartments (1). In addition to coat-dependent export of secretory cargo from the ER, retrieval (2) and retention (3) mechanisms operate to maintain overall compartmental organization.A related group of integral membrane proteins, referred to as the p24 family, are thought to act in concert with COPI and COPII to sort proteins during transport through the early secretory pathway. Initially identified on ER membranes (4) and subsequently detected as abundant proteins on COPI-and COPII-coated vesicles (5, 6), the function of p24 proteins in sorting remains unclear. In yeast strains lacking certain p24 members, some secretory proteins accumulate in the ER (e.g. invertase and the GPI-anchored protein Gas1p), while ER resident proteins that contain an HDEL retrieval motif are secreted and the unfolded protein response pathway is activated (6 -9). Based on these and other findings, the p24 proteins have been propo...

Localization of GDP-mannose transporter in the Golgi requires retrieval to the endoplasmic reticulum depending on its cytoplasmic tail and coatomer

“…This is consistent with the fact that the addition of C-terminal tags had not impaired the function and localization of GMT. As similar cases, it has been reported that the C-terminal peptides of Erv25p (KNYFKTKHII) and Rer1p (KYRYIPLDIGKKKYSHSSN), which also recycle between the ER and Golgi, bind to coatomer (Belden and Barlowe, 2001;Sato et al, 2001). Rer1p localizes to the Golgi at the steady state (Sato et al, 2001), like GMT.…”

Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

“…Appending the HA tag to the C terminus of full-length Erv26p also caused a shift in localization to Golgi membranes, although Erv26-HA displayed only mild reductions in pro-ALP transport and recognition by the COPII budding machinery. Based on these results, we conclude that the C-terminal tail of Erv26p contains COPI-and COPII-specific sorting signals that allow the protein to cycle between ER and Golgi compartments in accord with other shuttling cargo receptors (31,42,43). We suggest that removal of both transport signals in the tail deletion mutant (Erv26⌬C2-HA) produces a protein that is inefficiently packaged into COPII vesicles but ultimately traffics to the Golgi complex in a slower "bulk flow" manner.…”

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

“…As controls, cytoplasmic tail peptides of Erv25 were used. Under the conditions that reproduced the COPI binding to the Erv25 di-phenylalanine motif (YF in Erv25) (49), the Rrt6 tail peptide interacted with COPI in a manner dependent on the di-lysine motif (Fig. 7C).…”

“…COPI/COPII Binding to p24 C-tail Peptides-In vitro COPI binding assay of p24 cytoplasmic tail peptides was done as in Belden and Barlowe (49). Synthetic peptides used were Erv25C (CLKNYFKTKHII), Erv25YF-AA (CLKNAAKTKHII), Rrt6C (CTYLIIKIKSNPSSHIKKKGL), and Rrt6-KQ (CTYLIIKIK-SNPSSHIQQQGL).…”

“…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.…”