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Characterization of a novel sorting pathway at the Trans Golgi Network (TGN)

Final Report Summary - SORTMECH (Characterization of a novel sorting pathway at the Trans Golgi Network (TGN))

The Trans Golgi Network (TGN) is the central sorting station for newly synthesised proteins in the cell. Similar to a postal distribution center, incoming cargo is selected, sorted, and packaged to be carried to its final cellular destination by the postman in charge. Due to the involvement of numerous different cargoes and pleiomorhic carriers for the different destinations this process of protein sorting at the TGN is highly sophisticated. While the sorting event of lysosomal hydrolases at the TGN via mannose-6-phosphate receptor and clathrin-coated vesicles to the lysosome is well characterised, the relevant mechanism for other luminal proteins at the TGN remains elusive.

We recently discovered a new role for the actin severing protein twinstar in Drosophila and its orthologs in yeast (cof1) and mammalian cells (ADF and cofilin1) in regulation of luminal protein sorting at the TGN. This process is a novel calcium dependent sorting mechanism that directly links the actin cytoskeleton and ADF/cofilin to the TGN localized P-Type calcium ATPase SPCA1. The subordinate goal of the research group is to identify the mechanism of this process. Within the first funding period we have already published two novel major findings.

• SPCA1 contains ten transmembrane helices. N- and C-termini (N-term and C-term) are exposed to the cytosolic side as well as four inter-transmembrane domains, labeled L1 through L4. The N-term and L1 of SPCA1 fold to an actuator domain, while the largest cytosolic domain (L2) contains a nucleotide binding domain and a phosphorylation domain. Using purified proteins, we found that the P-domain of SPCA1 interacts with F-actin in a cofilin dependent manner. When expressed in HeLa cells, this domain inhibited Ca2+ entry into the TGN and caused missorting of secretory cargo. Furthermore, mutation of four amino acids in the mapped cofilin binding site of SPCA1, impaired Ca2+ import into the TGN and affected secretory cargo sorting.

• Binding of SPCA1 to cofilin and F-actin is required for the sorting reaction. Interestingly, cells depleted of ADF/cofilin or SPCA1 do not only missort secretory proteins but also release the soluble Golgi protein Cab45. Lodish and colleagues identified Cab45 as a Golgi-resident protein that is characterized by 6 Ca2+ binding EF hand domains. Consistent with this observation, we recently demonstrated that Cab45 localization to the Golgi is sensitive to Ca2+ levels, and that disrupting Golgi Ca2+ gradients induced Cab45 secretion by cells. Interestingly, knock down of Cab45 affected cargo sorting similar to the depletion of ADF/cofilin or SPCA1. In addition, Cab45 binds several secretory proteins in a Ca2+-dependent manner and this binding appears to be required for cargo sorting at the TGN.

• In addition, our new data show that Cab45 forms oligomers in the presence of Ca2+ in vitro and in living cells (Crevenna et al., 2016). Furthermore, Cab45 changes its secondary structure upon Ca2+ binding possibly to interact with its target proteins (Crevenna et al., 2016). Moreover, we observed that only the oligomeric form of Cab45 binds selectively to the specific cargo molecules COMP and LyzC but not to Cathepsin D in vitro (Crevenna et al., 2016). Finally 3D structural illumination microscopy (3D SIM) showed that Cab45, SPCA1 and cargo colocalize in specifc clusters at the TGN. We concluded from this data that Cab45 upon SPCA1-dependent Ca2+ influx into the lumen of the TGN, Cab45 binds Ca2+ triggering a conformational change and allowing oligomerization. These oligomers then bind specific proteins, thereby sorting cargo from non-cargo (Crevenna et al., 2016). We propose to refer to this Cab45 sorting oligomer as a cernosome, from the Latin cernere, which means to choose, sift, separate, decide or distinguish. The cernosome represents an elegant means of segregating different cargoes by recognition of the Cab45 oligomer. Thus we suggest that this is a unique way to export cargo molecules independent of a bona fide cargo recepor.