Abstract
The components of the COPII machinery, which are essential in establishing an effective Endoplasmic Reticulum (ER) to Golgi transport from ER exit sites (ERES), have been identified and characterized within the last 25 years. These consist of the essential Sec12, Sec23, Sec24, Sec13, Sec31 and Sar1 proteins. Together these components co‐operate in cargo‐selection as well as forming, loading and releasing budding vesicles from specific regions on the membrane surface of the ER. Coat components furthermore convey vesicle targeting towards the Golgi. However, not much is known about the mechanisms that regulate the COPII assembly at the vesicle bud site.
This thesis provides the first regulatory mechanism of COPII assembly in relation to ER‐membrane lipid‐signal recognition by the accessory protein p125A (Sec23IP).
The aim of the project was to characterize p125A function by dissecting two main domains in the protein; a putative lipid‐associating domain termed the DDHD domain that is defined by the four amino acid motif that gives the domain its name; and a ubiquitously found domain termed Sterile α‐motif (SAM), which is mostly associated with oligomerization and polymerization.
We first show, that the DDHD domain of p125A utilizes a stretch of positively charged residues (KGRKR) to bind lipid membranes that are enriched in Phosphatidylinositol‐4‐phosphates (PI(4)P). The specificity of the DDHD domain lipid recognition is demonstrated to be enhanced through p125A oligomerization mediated by the upstream SAM domain.
We then show that p125A is targeted specifically to ER exit sites (ERES) through a series of experiments where p125A expressing cells are incubated at lower temperatures. Incubation at either 15°C or 10°C inhibits cargo transport out of specific compartments that represent defined stages during the biosynthetic transport between the ER and the Golgi. We find that p125A associates predominantly with COPII‐marked ERES and dissociates from both the ER‐to Golgi‐intermediatecompartment (ERGIC) and from the cis‐Golgi compartment.
The same set of experiments also provides evidence that p125A functions at a later stage of the ER export. The temperature‐dependent block of ER export is shown to cause a clear segregation of ERES composed of Sec31A, Sec23 and p125A from the known COPII‐associating ERES nucleation scaffold protein mSec16A. The temperature block furthermore causes mSec16A to collect on the ER membrane in structures that neither co‐localize with ERGIC nor Golgi.
Using p125A double mutants that are impaired in lipid recognition, we show that the lipid recognizing activity of p125A regulates COPII organization. These double mutants are produced by introducing a point mutation (L690E) in the SAM domain that causes inhibition of its oligomerization, combined with either a charge reversal of the KGRKR lipid recognition motif within the DDHD domain (850(KGRKR/EGEEE)854 – DDHD‐PI‐X) or by deleting the entire DDHD domain (ΔDDHD). We demonstrate that p125A double mutants with defective lipid recognition strongly disperse ERES. This dispersal of the ERES can be rescued by replacing the DDHD with the PI(4)P recognizing Fapp1‐PH domain even if SAM(L690E) is still present in p125A. We additionally show that a stretch of cationic residues (KGRKR) in the DDHD abrogated p125A lipid recognition influences the proteins residency time at ERES.
Comparison of overexpressed of p125A wt, p125A(L690E)(PI‐X) and p125A(L690E)(ΔDDHD) with the expression of a GFP‐tagged mSec16A provides evidence that p125A lipid recognition furthermore promotes the displacement of COPII from the mSec16A scaffold during ERES assembly. The overexpression of p125A wt and p125A(L690E)(ΔDDHD), but not p125A(L690E)(PI‐X), causes p125A to aggregate in enlarged structures. The enlarged p125A wt structures show clear segregation from mSec16A, whereas the enlarged p125A(L690E)(ΔDDHD) structures become engulfed by the mSec16A. Surprisingly, no inhibition in the overall export of the temperature sensitive VSV‐G transport marker can be measured during these conditions.
Depletion of p125A by RNAi is additionally shown to cause perturbation of steady state level transport in HeLa cells. The transport perturbation manifests itself by the dispersion/shattering of the Golgi ribbon, where the Golgi instead appears to be broken into multiple mini‐stacks adjacent to ERES. The steady state transport level can be rescued by the introduction of an RNAi resistant p125A wt clone, but not by an RNAi resistant p125A double mutant.
These findings taken together point towards a model of p125A regulation at ERES, where p125A association with Sec31A, Sec23 and to specific ER membrane lipid signals provides linkage between the two COPII layers, and furthermore promotes displacement of the COPII cage from the mSec16A scaffold.
We additionally identify a structural fold termed WWE in the unstructured region of the p125A Nterminus that may potentially promote p125A binding to Sec31A.
We then further expand the temperature dependent ER export analysis of mSec16A to its smaller homolog mSec16B. Here, we examine mSec16B and mSec16A with regards to both proteins membrane targeting and association with ERES. We determine the localization of Sec16B by transient expression in HeLa cells, and find that the protein is evenly distributed throughout the cell except the nucleus at 37°C, as is also observed with mSec16A. When the temperature is lowered to 15°C, mSec16B mimics mSec16A further by associating and forming larger defined structures at the ER membrane that do not co‐localize with COPII, ERGIC53 or cis‐Golgi. Lowering the temperature further to 10°C, which arrests cargo at the ERES, maintains the formed structures substantially and decreases the even cellular distribution of mSec16B.
We further dissect both mSec16A and mSec16B, and show that the region in human mSec16B encompassing residues 35‐194 and the region in human mSec16A comprising residues 1096‐1190 maintain membrane binding irrespective of the removal of membrane associating proteins by salt wash or proteolytic digestion. However, neither mSec16B (35‐194) nor mSec16A (1096‐1190) maintain ERES targeting.
These findings support previous observations of the need for the membrane binding regions to be expressed in cis with a Central Conserved Domain (CCD) in both proteins to convey ERES targeting.
This thesis provides the first regulatory mechanism of COPII assembly in relation to ER‐membrane lipid‐signal recognition by the accessory protein p125A (Sec23IP).
The aim of the project was to characterize p125A function by dissecting two main domains in the protein; a putative lipid‐associating domain termed the DDHD domain that is defined by the four amino acid motif that gives the domain its name; and a ubiquitously found domain termed Sterile α‐motif (SAM), which is mostly associated with oligomerization and polymerization.
We first show, that the DDHD domain of p125A utilizes a stretch of positively charged residues (KGRKR) to bind lipid membranes that are enriched in Phosphatidylinositol‐4‐phosphates (PI(4)P). The specificity of the DDHD domain lipid recognition is demonstrated to be enhanced through p125A oligomerization mediated by the upstream SAM domain.
We then show that p125A is targeted specifically to ER exit sites (ERES) through a series of experiments where p125A expressing cells are incubated at lower temperatures. Incubation at either 15°C or 10°C inhibits cargo transport out of specific compartments that represent defined stages during the biosynthetic transport between the ER and the Golgi. We find that p125A associates predominantly with COPII‐marked ERES and dissociates from both the ER‐to Golgi‐intermediatecompartment (ERGIC) and from the cis‐Golgi compartment.
The same set of experiments also provides evidence that p125A functions at a later stage of the ER export. The temperature‐dependent block of ER export is shown to cause a clear segregation of ERES composed of Sec31A, Sec23 and p125A from the known COPII‐associating ERES nucleation scaffold protein mSec16A. The temperature block furthermore causes mSec16A to collect on the ER membrane in structures that neither co‐localize with ERGIC nor Golgi.
Using p125A double mutants that are impaired in lipid recognition, we show that the lipid recognizing activity of p125A regulates COPII organization. These double mutants are produced by introducing a point mutation (L690E) in the SAM domain that causes inhibition of its oligomerization, combined with either a charge reversal of the KGRKR lipid recognition motif within the DDHD domain (850(KGRKR/EGEEE)854 – DDHD‐PI‐X) or by deleting the entire DDHD domain (ΔDDHD). We demonstrate that p125A double mutants with defective lipid recognition strongly disperse ERES. This dispersal of the ERES can be rescued by replacing the DDHD with the PI(4)P recognizing Fapp1‐PH domain even if SAM(L690E) is still present in p125A. We additionally show that a stretch of cationic residues (KGRKR) in the DDHD abrogated p125A lipid recognition influences the proteins residency time at ERES.
Comparison of overexpressed of p125A wt, p125A(L690E)(PI‐X) and p125A(L690E)(ΔDDHD) with the expression of a GFP‐tagged mSec16A provides evidence that p125A lipid recognition furthermore promotes the displacement of COPII from the mSec16A scaffold during ERES assembly. The overexpression of p125A wt and p125A(L690E)(ΔDDHD), but not p125A(L690E)(PI‐X), causes p125A to aggregate in enlarged structures. The enlarged p125A wt structures show clear segregation from mSec16A, whereas the enlarged p125A(L690E)(ΔDDHD) structures become engulfed by the mSec16A. Surprisingly, no inhibition in the overall export of the temperature sensitive VSV‐G transport marker can be measured during these conditions.
Depletion of p125A by RNAi is additionally shown to cause perturbation of steady state level transport in HeLa cells. The transport perturbation manifests itself by the dispersion/shattering of the Golgi ribbon, where the Golgi instead appears to be broken into multiple mini‐stacks adjacent to ERES. The steady state transport level can be rescued by the introduction of an RNAi resistant p125A wt clone, but not by an RNAi resistant p125A double mutant.
These findings taken together point towards a model of p125A regulation at ERES, where p125A association with Sec31A, Sec23 and to specific ER membrane lipid signals provides linkage between the two COPII layers, and furthermore promotes displacement of the COPII cage from the mSec16A scaffold.
We additionally identify a structural fold termed WWE in the unstructured region of the p125A Nterminus that may potentially promote p125A binding to Sec31A.
We then further expand the temperature dependent ER export analysis of mSec16A to its smaller homolog mSec16B. Here, we examine mSec16B and mSec16A with regards to both proteins membrane targeting and association with ERES. We determine the localization of Sec16B by transient expression in HeLa cells, and find that the protein is evenly distributed throughout the cell except the nucleus at 37°C, as is also observed with mSec16A. When the temperature is lowered to 15°C, mSec16B mimics mSec16A further by associating and forming larger defined structures at the ER membrane that do not co‐localize with COPII, ERGIC53 or cis‐Golgi. Lowering the temperature further to 10°C, which arrests cargo at the ERES, maintains the formed structures substantially and decreases the even cellular distribution of mSec16B.
We further dissect both mSec16A and mSec16B, and show that the region in human mSec16B encompassing residues 35‐194 and the region in human mSec16A comprising residues 1096‐1190 maintain membrane binding irrespective of the removal of membrane associating proteins by salt wash or proteolytic digestion. However, neither mSec16B (35‐194) nor mSec16A (1096‐1190) maintain ERES targeting.
These findings support previous observations of the need for the membrane binding regions to be expressed in cis with a Central Conserved Domain (CCD) in both proteins to convey ERES targeting.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Number of pages | 182 |
| Publication status | Published - 2013 |