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Properties of a GTP sensitive microdomain in rough microsomes
Abstract:
Stripped rough microsomes (SRM) fuse when incubated with physiological concentrations of GTP and MgCl2. In order to examine further to what extent such fusions are associated with other membrane functions of rough endoplasmic reticulum, we have evaluated the role of cytosolically exposed peptide constituents of SRM in fusion, and the possible relationship of GTP/MgCl2-induced fusion in protein transport across endoplasmic reticulum (ER) membranes, and in ER-Golgi interactions. Controlled proteolytic digestion of SRM led to the loss of fusion capability at 15 micrograms/ml trypsin--a concentration which maintained the latency of intraluminal mannose-6-phosphatase. Hence, a cytosolically exposed protein(s) regulated fusion. Based on ribonuclease-induced ribosome capping experiments, it was further concluded that the cytosolic oriented protein(s) was sequestered beneath the ribosome. As co-translational cell free translocation of placental lactogen across SRM was similar in control membranes compared to those rendered incapable of fusing, it was concluded that the fusion phenomenon may not be related to translocation. Under conditions promoting homologous fusion of SRM or Golgi membranes, mixtures of the two membranes showed no heterologous membrane fusion as assessed morphologically or by the transport of newly synthesized membrane glycoprotein. These experiments attest to the specificity of cytosolically exposed protein(s) in regulating nucleotide/divalent cation-induced membrane fusion.
Insights
Cytosolic proteins regulate the fusion of stripped rough microsomes (SRM), a process essential for endoplasmic reticulum (ER) function. This fusion is distinct from protein transport and ER-Golgi interactions, highlighting specific regulatory mechanisms.
Area of Science:
- Cell Biology
- Molecular Biology
- Membrane Trafficking
Background:
- Stripped rough microsomes (SRM) exhibit fusion upon incubation with GTP and MgCl2.
- Understanding the regulation of membrane fusion is crucial for cellular processes like protein transport and organelle interaction.
Purpose of the Study:
- To investigate the role of cytosolically exposed proteins in SRM fusion.
- To determine the relationship between GTP/MgCl2-induced fusion and protein transport across ER membranes.
- To examine the involvement of SRM fusion in ER-Golgi interactions.
Main Methods:
- Controlled proteolytic digestion of SRM using trypsin.
- Ribonuclease-induced ribosome capping experiments.
- Co-translational cell-free translocation assays.
- Morphological assessment and glycoprotein transport analysis of membrane fusion.
Main Results:
- Proteolytic digestion at low trypsin concentrations abolished SRM fusion while preserving intraluminal enzyme latency, indicating a role for cytosolically exposed proteins.
- Ribosome capping experiments suggested these regulatory proteins are located beneath the ribosome.
- SRM fusion capability did not affect co-translational protein translocation.
- No heterologous fusion occurred between SRM and Golgi membranes, demonstrating fusion specificity.
Conclusions:
- Cytosolically exposed proteins, sequestered beneath ribosomes, specifically regulate nucleotide/divalent cation-induced membrane fusion in SRM.
- SRM fusion is mechanistically distinct from protein translocation across the ER membrane.
- The fusion process is specific and does not extend to heterologous interactions with Golgi membranes.