KDEL and KKXX Retrieval Signals Appended to the Same Reporter Protein Determine Different Trafficking between Endoplasmic Reticulum, Intermediate Compartment, and Golgi Complex

Abstract

Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, where O-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms.

Nef Binds p6* in GagPol during Replication of Human Immunodeficiency Virus Type 1

“…As expected, a perinuclear subpopulation of the CD8.NefNL4-3 chimera localized partially to the TGN ( were not detected at ER structures (data not shown). This pattern is reminiscent of that described in a recent report, where a CD8.E19 chimera that contained the KKMP motif at the C terminus was localized mostly to the intermediate compartment and the TGN (27,45). We conclude that the mutant CD8.NefKKXX and the CD8.NefF12 chimeras both localized to the intermediate compartment in cells.…”

Lower levels of surface B-cell-receptor expression in chronic lymphocytic leukemia are associated with glycosylation and folding defects of the μ and CD79a chains

“…However, the colocalization of these molecules with calnexin suggests that they may have been misfolded or may have undergone retrograde transport to the ER from the trans-Golgi compartment. 19,20 Some colocalization of chains with cis-Golgi markers was observed, but only in patients with higher levels of surface IgM expression, consistent with this possibility (data not shown). Nevertheless, the possible involvement of the N-linked glycosylation of both and CD79a chains in defective surface expression of IgM in CLL is of particular interest.…”

The cytosolic chaperone α-Crystallin B rescues appropriate folding and compartmentalization of misfolded multispan transmembrane proteins

“…To perform the PLA analysis we started with human hepatoma Huh-7 cells. These cells are larger and flatter than HEK293 cells, exhibit a well-defined and extended ER when observed with light microscopy (D'Agostino et al, 2011; Stornaiuolo et al, 2003), and do not express a detectable level of CRYAB (data not shown). Next, we generated a PLA-suitable mouse polyclonal antibody, specific for the cytosolic C-terminal tail of Fz4-FEVR.…”