Endoplasmic Reticulum Calcium Regulates the Retrotranslocation of Trypanosoma Cruzi Calreticulin to the Cytosol

Abstract

For most secretory pathway proteins, crossing the endoplasmic reticulum (ER) membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD), is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT) being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C), since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.

Structural Features Affecting Trafficking, Processing, and Secretion of Trypanosoma cruzi Mucins

“…Experiments aimed at quantifying the association of GPI-minus reporters with BiP and/or other molecular chaperones may shed some light on this issue. In this line, however, it is worth noting that calreticulin, the main N-glycoprotein chaperone in T. cruzi (35), does not seem to play a significant role in apo-mucin maturation/trafficking, as revealed by the A66 mutant. Further evidence weigh against "quality control" as a mechanism for regulating export of ⌬GPI reporters.…”

“…As for the molecular basis underlying this observation, two alternative hypotheses can be envisaged. On one hand, it is possible that ⌬GPI reporters, which are normally membrane bound, are unable to attain a stable conformation when expressed in soluble form and are thus retained in the ER by folding quality control systems (35). Experiments aimed at quantifying the association of GPI-minus reporters with BiP and/or other molecular chaperones may shed some light on this issue.…”

“…Following centrifugation for 10 min at 15,000 ϫ g, the supernatant (cytosolic fraction) was removed and the pellet was resuspended in the same buffer supplemented with 1% Nonidet P40 (Nonidet P-40). The suspension was centrifuged as above and the supernatant (microsomal fraction) was collected (35). Aliquots were fractionated in 10 -15% SDS-PAGE, transferred to PVDF membranes (GE Healthcare), and analyzed by Western blot using mAb anti-FLAG (clone M2, Sigma) at 1:5,000 dilution followed by HRP-conjugated secondary antibodies (Sigma) and chemiluminescence substrate (Pierce).…”

Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation

“…Afshar et al (24) claimed that cyt-CRT is not a proteasomal substrate because it did not detect ubiquitinated cyt-CRT in cells. On the other hand, Labriola et al (25) suggested that CRT is degraded in the cytosol by proteasome because, after exposure of cells to an inhibitor of ER Ca 2ϩ -ATPase, cyt-CRT level was reduced by CHX treatment and increased by MG132 treatment. Our present findings indicate that both CRT and R-CRT are proteasomal substrates, although the degradation rate of R-CRT is lower than that of CRT (Figs.…”

“…However, a significant subset of proteins displays ubiquitination-independent turnover (21), which is not surprising in view of the co-existence of several types of proteasomal complexes in eukaryotic cells (22). The possible role of CRT as a substrate for proteasomal degradation is controversial (23)(24)(25). The degradation mechanism of R-CRT remains unknown.…”