Question

Consider cargo proteins that are destined for secretion to the extracellular space. Please predict where the cargo will end up (ex. in the cytoplasm, a specific organelle, transport vesicles, multiple places, extracellular space) for: (i) & (ii) Each individual condition; & (iii) The combination of both conditions.

A. (i) Normal Sar1 is mutated so it binds tightly to GDP and cannot exchange it for GTP. (This leads to Sar1-GDP being the only form of the protein in the cell.)

(ii) Normal ARF1 is mutated so it binds tightly to GDP and cannot exchange it for GTP. (This leads to ARF1-GDP being the only form of the protein in the cell.)

(iii) Both

Answer 1

i) Secreted after translation and entry into ER lumen will be recruited to the ER membrane exit sites by cargo capture or bulk flow. ER exit sites lack ribosomes. Newly synthesized proteins are transported from the ER to Golgi by COPII coated vesicles that contain Sar1 protein. For formation of these vesicles, Sar1 should be activated. This activation requires a guanine-nucleotide exchange factor (GEF) Sec12. Sar1-GDP is soluble and will become membrane bound when the membrane bound Sec12 GEF. As a result Sar1 will exchange GDP for GTP. This will expose a fatty acid chain which allows the Sar1 to embed into ER membrane. As a result, a positive curvature is induced. Once Sar1 is activates, it will recruit two proteins heterodimers of Sec 23 and Sec24. Sec23/Sec24 will bind to Sar1-GTP and stabilize the positive curvature. Sec 23-Sec24 heterodimers will then form a complex with cargo protein. This complex is called the pre-budding complex. Once the pre-budding complex is formed, there is binding of Sec13-Sec 31 to Sec 23-Sec24. This interaction enhances the activity of GAP (GTPase activating protein) of Sec23. As a result, the coat assembly is completed. The GTPase activity of Sar1 is responsible for the converting Sar1-GTP to Sar 1-GDP and release of the COPII coated vesicles.

If Sar1-GDP cannot be exchanged for Sar-1 GTP, then Sar-1 is not activated. Hence, it cannot recruit Sec23/24 and this will inhibit coat assembly and therefore no formation of COPII coated vesicles. Thus, the proteins cannot exit the ER and will remain in the ER membrane exit sites.

ii) COPI coated vesicles are involved in transport of cargo protein from cis end of Golgi back to the ER (retrograde transport). They are also involved in intra-Golgi transport. Retrograde transport is required for sending escaping proteins reaching the Golgi back to ER. ARF1 (ADP-ribosylation factor 1) is required for transport of protein from Golgi in COP1 coated vesicles. It is involved in vesicle budding and coating in the Golgi complex. ARF1 is localized in Golgi in active form of ARF1-GDP. It is then recruited to membrane when GTP binds to it (via its GBF/Gea or BIG/Sec7 GEF), as an N-myristoylated amphipathic helix is exposed, helping in membrane anchoring. This GTP activation is necessary for recruitment of COPI coat protein via association with clathrin adapter complexes AP-1. Coat release occurs only after GTP hydrolysis to form GDP-ARF1.

If ARF1 is mutated, such that it cannot be activated by GTP binding, ARF1 will be present in inactive soluble form in Golgi lumen. Hence, there is no formation of the COPI vesicle and the cargo protein. The escaped cargo protein remains in the cis-side of Golgi complex and is not transported to the ER. ARF1 transport is also required for intra Golgi transport. Hence, no proteins will be transported out of the Golgi if only ARFI-GDP is present. The cargo proteins will hence, remain in the Golgi apparatus.

iii) COPII coated vesicles are crucial for transport of all newly synthesized cargo proteins destined for extracellular space. If COPII coated vesicles are not formed, then the cargo protein will not reach the Golgi and remain in ER. As SAR-1 is in GDP bound state, no COPII vesicles are formed. Thus, the protein remains in the ER membrane. No clathrin coated secretory vesicles will be formed as there is no transport to Golgi. The absence of ARFI-GTP will have not much effect as ARF1-GTP is only required for retrograde Golgi to ER transport or intra-Golgi transport. Thus, the cargo proteins should be seen only in the ER.