Eason ZengSimon Xu

Bubbles Zhang

Plant Retromer, Localized to the Prevacuolar Compartmentand Microvesicles in Arabidopsis, May Interact with Vacuolar

Sorting Receptors

•What is retromer

•Where is retromer

•What is the function of retromer

•What is the structure of retromer

•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.

The function is :

Recycle the receptor

• 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

The structure and function of each subunit of the complex.

Let’s take the example of the retromer in mammalian cells.

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.”

Your Name: Simon Xu

Your Teacher: PHD. Yin

Your Grade: Junior

Place photo here

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.

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

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.

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

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

Structure of the VPS29–VPS35 subcomplex

Structure of the VPS29–VPS35 subcomplex

Structure of the VPS29–VPS35 subcomplex

Working model for the assembly and function of mammalian retromer

Retromer in plant

Retromer in plant?

you kidding?Whether plant cells express a retormer-like complex and where the complex is

localized?

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

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

Other experimental methods and results

Let’s welcome

Bubbles!!!

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 whichretromer associates as being the PVC

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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

Figure 2. Reactivity of Retromer Antibodies and Localization of Retromer Proteins in Subcellular Fractions of Arabidopsis and Tobacco BY-2 Cells.

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

Distribution of Retromer Proteins in Subcellular Fractions

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.

• 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.

200KD 145KD 66kD

VPS35 (89kD)

Fractions

VPS29 (21kD)

VPS26 (35kD)

Figure 4. Superdex 200 Column Separation of a Salt-Dissociated Arabidopsis P100 Membrane Extract.

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。

Retromer Binds to PVC Membranes and Possibly to Microvesicles

Figure 5. Sedimentation Characteristics of Retromer Binding Membranes in Sucrose Gradients.

Retromer Binds to PVC Membranes and Possibly to Microvesicles

Figure 6. Negative Contrast Immunographs of High-Density Sucrose Fractions (Bars =100 nm ).

overview

VPS35 and VPS26 immunogold

clathrin-coated vesicles

retromer-coated vesicles

Retromer Binds to PVC Membranes and Possibly to Microvesicles

Figure 6. Negative Contrast Immunographs of High-Density Sucrose Fractions (Bars =100 nm ).

high-magnification detail

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

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

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

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

Wortmannin Treatment Separates VPS35 and VSRAt-1

Figure 9. Wortmannin Alters the Distribution of Retromer in Tobacco BY-2 Cells.

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.

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).

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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).

3”R”s

Reduce

Reuse

Recycle

retromer

That’s all! Thank you!

Let’s have a happy lunch!