Membrane Morphology and Function
The trials and tubule-ations of Rab6 involvement in Golgi-to-ER retrograde transport Linda F. Heffernan* and Jeremy C. Simpson*1 *School of Biology and Environmental Science & Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Belfield, Dublin 4, Ireland
Abstract In the early secretory pathway, membrane flow in the anterograde direction from the endoplasmic reticulum (ER) to the Golgi complex needs to be tightly co-ordinated with retrograde flow to maintain the size, composition and functionality of these two organelles. At least two mechanisms of transport move material in the retrograde direction: one regulated by the cytoplasmic coatomer protein I complex (COPI), and a second COPI-independent pathway utilizing the small GTP-binding protein Rab6. Although the COPI-independent pathway was discovered 15 years ago, it remains relatively poorly characterized, with only a handful of machinery molecules associated with its operation. One feature that makes this pathway somewhat unusual, and potentially difficult to study, is that the transport carriers predominantly seem to be tubular rather than vesicular in nature. This suggests that the regulatory machinery is likely to be different from that associated with vesicular transport pathways controlled by conventional coat complexes. In the present mini-review, we have highlighted the key experiments that have characterized this transport pathway so far and also have discussed the challenges that lie ahead with respect to its further characterization.
Introduction The cytoplasm of eukaryotic cells consists of a wide variety of membrane-bound organelles each carrying out specific functions. Membrane traffic pathways between these organelles play key roles in transporting protein and lipid cargo. Traditionally, they are subdivided into the anterograde or secretory pathway and the endocytic pathway, which can also include the retrograde pathway operating between the Golgi complex and the endoplasmic reticulum (ER). In the secretory pathway, newly synthesized material is transported from the ER through the Golgi complex to the endosomal system, the lysosomes or the cell surface [1]. This anterograde flow is counterbalanced by the constant recycling of proteins and lipids, ensuring the steady-state distributions of residents and the overall composition and architecture of the organelles themselves. In addition to recycling events between individual Golgi cisternae [2], Golgi glycosylation enzymes can also recycle in a retrograde manner back to the ER. Fluorescence microscopy approaches, specifically using photobleaching and a transfected galactose transferase (GalTase)–GFP construct (as a representative of Golgi glycosylation enzymes), have estimated that 30 % of Golgiresident enzymes can be in the ER at steady-state in HeLa, Chinese-hamster ovary (CHO), normal rat kidney (NRK), Key words: Golgi complex, membrane traffic, Rab6, Rab GTPase, retrograde transport. Abbreviations: ARF1, ADP-ribosylation factor 1; BFA, brefeldin A; BicD, Bicaudal-D; COPI, coatomer protein I; ER, endoplasmic reticulum; GalTase, galactose transferase; GBF1, Golgispecific BFA-resistant factor 1; GEF, guanine-nucleotide-exchange factor; LPAT, lysophospholipid acyltransferase; LPL, lysophospholipid; PLA, phospholipase A; STx, Shiga toxin; STxB, B-chain from STx; TGN, trans-Golgi network. 1 To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2014) 42, 1453–1459; doi:10.1042/BST20140178
COS and PTK1 cells [3]. These results were shown not to be an artefact from the overexpression approach taken, and the detected fluorescence in the ER was not due to synthesis of new GalTase–GFP, as was evident from experiments in cycloheximide-treated cells. This pool of Golgi-resident enzymes found in the ER was shown to originate in the Golgi, as during the recovery of fluorescence in the ER following photobleaching, the amount of fluorescence in the Golgi decreased proportionately. Quantitative measurements indicated that steady-state distributions in the Golgi and ER could be re-established within ∼5 h [3]. Other studies attempting to quantify the levels of Golgi proteins present in the ER have used cytoplasts as a model system. Although such approaches have estimated a lower percentage of Golgi residents in the ER at any particular time (
The trials and tubule-ations of Rab6 involvement in Golgi-to-ER retrograde transport Linda F. Heffernan* and Jeremy C. Simpson*1 *School of Biology and Environmental Science & Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Belfield, Dublin 4, Ireland
Abstract In the early secretory pathway, membrane flow in the anterograde direction from the endoplasmic reticulum (ER) to the Golgi complex needs to be tightly co-ordinated with retrograde flow to maintain the size, composition and functionality of these two organelles. At least two mechanisms of transport move material in the retrograde direction: one regulated by the cytoplasmic coatomer protein I complex (COPI), and a second COPI-independent pathway utilizing the small GTP-binding protein Rab6. Although the COPI-independent pathway was discovered 15 years ago, it remains relatively poorly characterized, with only a handful of machinery molecules associated with its operation. One feature that makes this pathway somewhat unusual, and potentially difficult to study, is that the transport carriers predominantly seem to be tubular rather than vesicular in nature. This suggests that the regulatory machinery is likely to be different from that associated with vesicular transport pathways controlled by conventional coat complexes. In the present mini-review, we have highlighted the key experiments that have characterized this transport pathway so far and also have discussed the challenges that lie ahead with respect to its further characterization.
Introduction The cytoplasm of eukaryotic cells consists of a wide variety of membrane-bound organelles each carrying out specific functions. Membrane traffic pathways between these organelles play key roles in transporting protein and lipid cargo. Traditionally, they are subdivided into the anterograde or secretory pathway and the endocytic pathway, which can also include the retrograde pathway operating between the Golgi complex and the endoplasmic reticulum (ER). In the secretory pathway, newly synthesized material is transported from the ER through the Golgi complex to the endosomal system, the lysosomes or the cell surface [1]. This anterograde flow is counterbalanced by the constant recycling of proteins and lipids, ensuring the steady-state distributions of residents and the overall composition and architecture of the organelles themselves. In addition to recycling events between individual Golgi cisternae [2], Golgi glycosylation enzymes can also recycle in a retrograde manner back to the ER. Fluorescence microscopy approaches, specifically using photobleaching and a transfected galactose transferase (GalTase)–GFP construct (as a representative of Golgi glycosylation enzymes), have estimated that 30 % of Golgiresident enzymes can be in the ER at steady-state in HeLa, Chinese-hamster ovary (CHO), normal rat kidney (NRK), Key words: Golgi complex, membrane traffic, Rab6, Rab GTPase, retrograde transport. Abbreviations: ARF1, ADP-ribosylation factor 1; BFA, brefeldin A; BicD, Bicaudal-D; COPI, coatomer protein I; ER, endoplasmic reticulum; GalTase, galactose transferase; GBF1, Golgispecific BFA-resistant factor 1; GEF, guanine-nucleotide-exchange factor; LPAT, lysophospholipid acyltransferase; LPL, lysophospholipid; PLA, phospholipase A; STx, Shiga toxin; STxB, B-chain from STx; TGN, trans-Golgi network. 1 To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2014) 42, 1453–1459; doi:10.1042/BST20140178
COS and PTK1 cells [3]. These results were shown not to be an artefact from the overexpression approach taken, and the detected fluorescence in the ER was not due to synthesis of new GalTase–GFP, as was evident from experiments in cycloheximide-treated cells. This pool of Golgi-resident enzymes found in the ER was shown to originate in the Golgi, as during the recovery of fluorescence in the ER following photobleaching, the amount of fluorescence in the Golgi decreased proportionately. Quantitative measurements indicated that steady-state distributions in the Golgi and ER could be re-established within ∼5 h [3]. Other studies attempting to quantify the levels of Golgi proteins present in the ER have used cytoplasts as a model system. Although such approaches have estimated a lower percentage of Golgi residents in the ER at any particular time (
