1. RETROMER Eason Zeng Simon Xu Bubbles Zhang
Plant Retromer, Localized to the Prevacuolar Compartment
and Microvesicles in Arabidopsis, May Interact with Vacuolar
Sorting Receptors
Eason Zeng
Simon Xu
Bubbles Zhang

2. What is retromer
Where is retromer
What is the function of retromer
What is the structure of retromer

3. Definition
Retromer is a complex of Vacuolar protein sorting proteins that mediate retrieval of lysosomal hydrolases（溶酶体水解酶） from endosomes to the trans（反） golgi network.

5. The function is :
Recycle the receptor

6. The retromer complex was identified and deeply studied in yeast 、plants and mammals.
The retromer complex consists of 5 proteins (subunit)
in yeast: vps35p,vps26p,vps29p,
vps17p,vps5p
In mammals  : Vps35, Vps26, Vps29,
SNX1,SNX2
in plant: vps35p,vps26p,vps29p,
SNX2（？）,vps5p

7. The structure and function of each subunit of the complex.
Let’s take the example of the retromer in mammalian cells.

8. Your Name: Simon Xu Your Teacher: PHD. Yin Your Grade: Junior
Place photo here
The Structures of retromer models in yeasty, mammalian and plant cells.
“When you are willing to make sacrifices for a great cause, you will never be alone.”

9. The Retromer model in yeasty cells
Two sub complexes
Vps5p and Vps17p have a structural role
Vps35p, Vps29p, Vps26p are for cargo selections
Vps5p and Vps17p both contain BAR domains in their C-terminal regions.
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10. The Retromer model in yeasty cells
Vps5p and Vps17p both have a PX domain that binds to phosphatidylinositol-3-phosphate (PtdIns3P) （三磷酸磷脂酰环己六醇）
Vps10 and MPR are cargo receptors

11. Details in functions of domains in yeasts
Vps35p, Vps29p, Vps26p are required for localization of Vps10p
Vps5p and Vps17p functions in maintaining the proper localization of Vps10.
PtdIns3P is required for the proper localization of Vps5p-Vps17p.
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12. Details in functions of domains in yeasts
Vps35p interacts directly with the Vps10p
Vps26p interacts with the membrane as a linker
Vps5-Vps17p dimer provides the mechanical impetus to bud a vesicle
BAR domain can induce the tubulation
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13. How about the retromer in mammalian cells
Vps5, Vps17 turn to SNX1and SNX2 (the status of SNX2 as a subunit of retromer is unclear )
SNX1 and SNX2 may form heterodimers or homodimers that bind to PtdIns3P-enriched endosomes
Vps29p functions as a linker between Vps35p and the Vps17p/Vps5p dimer
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14. Structure of the VPS29–VPS35 subcomplex

15. Structure of the VPS29–VPS35 subcomplex

16. Structure of the VPS29–VPS35 subcomplex

17. Working model for the assembly and function of mammalian retromer
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18. Retromer in plant? you kidding?
Whether plant cells express a retormer-like complex and where the complex is localized?

19. Plant retromer HOMOLOGS
Three isoforms for vps35,one for vps29, two for vps26 and three for vps5.
Identity ranged between 25%(vps35)and 51%(vps26)
A sequence corresponding to vps17could not be found
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20. Plant Retromer HOMOLOGS
the VPS26 antiserum(抗血清）
did we observe a cross-reaction with the corresponding
Yeast Vps35p antibodies did not recognize any polypeptide in plant extracts

21. Other experimental methods and results
Let’s welcome
Bubbles!!!

22. Antibody (to identify the complex and combine）
Clone ↓
Make the complex ↓
Antibody (to identify the complex and combine） ↓
Immunofluorescence confocal (免疫荧光共聚焦）microscopy and
Immunogold（免疫金颗粒） electron microscopy ↓
identified the organelle with which
retromer associates as being the PVC

23. RESULTS
Identification of Plant Retromer Homologs and Cross-Reactivity of Antisera
Distribution of Retromer Proteins in Subcellular Fractions
Solubilized VPS35, VPS29, and VPS26 Remain Together as a Subcomplex
Retromer Binds to PVC Membranes and Possibly to Microvesicles
Retromer Localizes to Multivesicular Bodies/PVC in Situ
Wortmannin Treatment Separates VPS35 and VSRAt-1
VSRAt-1 Is Immunoprecipitated with VPS35 Antibodies
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24. Distribution of Retromer Proteins in Subcellular Fractions
VPS35 and VPS29 are both present primarily in membrane fractions.
By contrast, considerably more VPS26 antigen was detected in the S100 cytosol fractions.
Pellets: P13 and P100
S100 supernatant
Figure 2. Reactivity of Retromer Antibodies and Localization of Retromer Proteins in Subcellular Fractions of Arabidopsis and Tobacco BY-2 Cells.
After sequential centrifugations filtered homogenates of Arabidopsis and tobacco BY-2 cells. They got The three fractions——P13 and P100 pellets and S100 supernatant. And then the three fractions were subjected to protein gel blotting with VPS35, VPS29, and VPS26 antibodies. As we can see from this figure, VPS35 and VPS29 are both present primarily in membrane fractions. By contrast, more more VPS26 antigen was detected in the S100 than in the two membrane fractions. The similar phenomenon were happened in the yeast’s subunits.

25. Distribution of Retromer Proteins in Subcellular Fractions
Why did this happen? They consider this from the subunits function. VPS35 recruits the receptor and VPS29 oligomerizes the Vps35p/Vps10p complexes into budding domains.

26. Solubilized VPS35, VPS29, and VPS26 Remain Together as a Subcomplex
the P100 pellet of Arabidopsis was resuspended(重悬浮) in different solutions as shown in Figure 3.
Figure 3. Dissociation of the Large Retromer Subunit from Arabidopsis P100 Membranes.
200KD 145KD kD
VPS35 (89kD)
Fractions
VPS29 (21kD)
VPS26 (35kD)
Figure 4. Superdex 200 Column Separation of a Salt-Dissociated Arabidopsis P100 Membrane Extract.
All three retromer proteins were detected in a fraction with a size of about 150 kD. these three retromer components remain together upon salt-induced dissociation from the membrane surface.
the P100 pellet of Arabidopsis was resuspended in different solutions as shown in Figure 3. A substantial proportion of VPS35 antigen dissociated from the membranes at low salt (250 mM NaCl) concentrations. Complete release and solubilization can be achieved with either 2M urea or 1% Triton X-100 treatments. The solubilized proteins separated on a Superdex 200 column. Fractions (0.5 mL) were collected and the proteins precipitated and subjected to protein gel blotting with VPS35, VPS29, and VPS26 antisera. All three retromer proteins were detected in a fraction with a size of about 150 kD. This corresponds to the sum of these three retromer components and indicates that they remain together upon salt-induced dissociation from the membrane surface.

27. Retromer Binds to PVC Membranes and Possibly to Microvesicles
linear sucrose density gradients
isopycnic centrifugations(等密度离心)
原理：当不同颗粒存在浮力密度差时，在离心力场下，在密度梯度介质中，颗粒或向下沉降，或向上浮起，一直移动到与它们各自的密度恰好相等的位置，在这里颗粒没有重量，不管离心多长时间，它们再也不移动了，形成一系列密度区。从而使不同浮力密度的物质得到分离。
To obtain evidence on the identity of the retromer binding membranes, we separated performed P100 membranes from Arabidopsis and BY-2 cells by isopycnic centrifugations(等密度离心) on linear sucrose density gradients（线性蔗糖密度梯度).

28. Retromer Binds to PVC Membranes and Possibly to Microvesicles
VPS35 collects in two regions of the gradient: a major peak around 47 to 52% sucrose and a broad band between 30 and 42% sucrose,(单击) which corresponds to the densities of VSRAt-1 which is vacuolar sorting receptor VSRAt-1 and PEP12(SYP21)-bearing membranes which is PVC’s markers. The VPS35 profiles for BY-2 cells are less clearly depicted into two separate peaks than for Arabidopsis but cover the same stretch of the gradient..
Figure 5. Sedimentation Characteristics of Retromer Binding Membranes in Sucrose Gradients.

29. Retromer Binds to PVC Membranes and Possibly to Microvesicles
overview
clathrin-coated vesicles
VPS35 and
VPS26 immunogold
retromer-coated
vesicles
Because the majority of plant membranes, including the plasma membrane, have equilibrium densities less than 44% sucrose, they decided to examine the composition of the high-density VPS35-containing fractions by negative staining. They found this fraction contained clearly identifiable clathrin-coated vesicles(The arrows point to clathrin-coated vesicles) but also a population of fuzzy-coat structures, which at a diameter of 90 nm(nanometer) were slightly larger than the clathrin-coated vesicles. (单击)As the C part of this figure shows these structures reacted positively toward VPS35 and VPS26 antibodies in immunogold negative staining (Figure 6C), They tentatively claim to have identified retromer-coated vesicles(the arrowheads label putative retromer-coated vesicles).
What if the fuzzy-coat structures are COPII and COPI vesicles. They used coat proteins’ antibodies of the COPII and COPI vesicles to label the structures, but the failed.
Figure 6. Negative Contrast Immunographs of High-Density Sucrose Fractions (Bars =100 nm ).

30. Retromer Binds to PVC Membranes and Possibly to Microvesicles
high-magnification detail
Figure 6. Negative Contrast Immunographs of High-Density Sucrose Fractions (Bars =100 nm ).

31. Retromer Localizes to Multivesicular Bodies/PVC in Situ
although there is hardly any colocalization between VPS35 and the Golgi marker, a substantial colocalization was registered with the PVC SNARE PEP12 Exactly the same combination of labeling data was obtained when the distribution of VPS35 was compared with VSR by double antibody staining
They used immunofluorescence microscopy(荧光显微镜) to identify where the VPS35, VPS29, and VPS26 antibodies colocalize with the Golgi markers or the PVC markerss.
This is convincingly underscored by a statistical analysis of the labeling results, with only ;2% of the retromer label associated with the Golgi apparatus in comparison with >80% with the PVC markers.
With labeling conditions adjusted to minimize unspecific background staining, gold particles were almost exclusively restricted to multivesicular bodies (MVBs; Figure 8) Gold label was found on or near the boundary membrane of the MVB, with little present on the interior vesicles.

32. Retromer Localizes to Multivesicular Bodies/PVC in Situ(原位)
A good colocalization of VPS26 with VSR was also obtained.
high degree of overlap in the images obtained with
VPS26 against VPS29 and PS29 against VPS35
They used immunofluorescence microscopy(荧光显微镜) to identify where the VPS35, VPS29, and VPS26 antibodies colocalize with the Golgi markers or the PVC markerss.
This is convincingly underscored by a statistical analysis of the labeling results, with only ;2% of the retromer label associated with the Golgi apparatus in comparison with >80% with the PVC markers.
With labeling conditions adjusted to minimize unspecific background staining, gold particles were almost exclusively restricted to multivesicular bodies (MVBs; Figure 8) Gold label was found on or near the boundary membrane of the MVB, with little present on the interior vesicles.

33. Retromer Localizes to Multivesicular Bodies/PVC in Situ
only about 2% of the retromer label associated with the Golgi apparatus in comparison with >80% with the PVC markers

34. Figure 8. Immunogold Localization of Retromer to MVBs in Tobacco BY-2 Cells.

35. Wortmannin Treatment Separates VPS35 and VSRAt-1
To determine whether the signals for VSRAt-1 and VPS35 remained colocalized on the PVC,wetherefore performed immunofluorescence microscopy on BY-2 cells treated with a concentration of wortmannin (16 mM) that was known to cause changes in PVC size and morphology.
This observation suggested wortmannin was causing the large, VPS35-containing retromer subunit to be displaced from the surface of the PVC.
We therefore isolated S100 and P100 fractions from control- and wortmannin-treated Arabidopsis and BY-2 cells and probed protein gel blots with VPS35 antibodies (Figure 10). However, at all times tested (from 20 min to 6 h), there was no significant difference in the relative signal strengths for cytosolic and membrane-bound VPS35 antigen between control and drug-treated samples (Figure 10). This indicates that wortmannin did not cause the release of VPS35 from the surface of the MVB. These protein gel blots were also performed with anti-VPS29 and anti-VPS26 with very similar results (data not shown). We surmised that the separation of the VSRAt-1 and VPS35 signals reflected their spatial redistribution to different domains in the membrane of the PVC.
Figure 9. Wortmannin Alters the Distribution of Retromer in Tobacco BY-2 Cells.

36. Wortmannin Treatment Separates VPS35 and VSRAt-1
Figure 10. Wortmannin Does Not Lead to a Release of the Large Subunit of Retromer into the Cytosol.

37. VSRAt-1 Is Immunoprecipitated with VPS35 Antibodies
immunoprecipitated VPS35 (Figure11A, lane 1) and an antigen reacting with the VSRAt-1 antibodies (Figure 11A, lane 6).
controls .
withVPS35 antibodies, a signal (arrow) was detected at the expected size ( ～90 kD).
with VSRAt-1 antibodies, and a signal (arrow) was seen at the expected size (～ 80 kD).
Using VPS35 antisera covalently coupled to protein-A Sepharose beads, we were able to immunoprecipitate VPS35 (Figure 11A, lane 1) and an antigen reacting with the VSRAt-1 antibodies (Figure 11A, lane 6). The protein source for these experiments was P100 membranes from the dense fractions (48 to 51% w/w) of the Arabidopsis linear sucrose gradients.
PI= 等电点
From lane2 to lane5 are different tyes of control immunoprecipitations.
After protein gel blot transfer of proteins in the 2D gels, membranes were probed withVPS35antibodies andthenwashedandreprobed
(without stripping) with VSRAt-1 antibodies

38. 3”R”s
Reduce
Reuse
Recycle
retromer

39. Let’s have a happy lunch!
That’s all!
Thank you!
Let’s have a happy lunch!